Stabilized bioactive peptides and methods of identification, synthesis and use

ABSTRACT

An intracellular selection system allows concurrent screening for peptide bioactivity and stability. Randomized recombinant peptides are screened for bioactivity in a tightly regulated expression system, preferably derived from the wild-type lac operon. Bioactive peptides thus identified are inherently protease- and peptidase-resistant. Also provided are bioactive peptides stabilized by a stabilizing group at either the N-terminus, the C-terminus, or both. The stabilizing group can take the form of a small stable protein, such as the Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, or glutathione reductase, or one or more proline residues.

[0001] This patent application claims the benefit of U.S. Provisional Patent Applications Serial Nos. 60/104,013, filed 13 Oct. 1998, and 60/112,150, filed 14 Dec. 1998.

BACKGROUND OF THE INVENTION

[0002] Bioactive peptides are small peptides that elicit a biological activity. Since the discovery of secretin in 1902, over 500 of these peptides which average 20 amino acids in size have been identified and characterized. They have been isolated from a variety of systems, exhibit a wide range of actions, and have been utilized as therapeutic agents in the field of medicine and as diagnostic tools in both basic and applied research. Tables 1 and 2 list some of the best known bioactive peptides. TABLE 1 Bioactive peptides utilized in medicine Size In Amino Name Isolated From Acids Therapeutic Use Angiotensin II Human Plasma 8 Vasoconstrictor Bradykinin Human Plasma 9 Vasodilator Caerulein Frog Skin 10 Choleretic Agent Calcitonin Human Parathyroid 32 Calcium Gland Regulator Cholecystokinin Porcine Intestine 33 Choleretic Agent Corticotropin Porcine Pituitary 39 Hormone Gland Eledoisin Octopod Venom 11 Hypotensive Agent Gastrin Porcine Stomach 17 Gastric Activator Glucagon Porcine Pancreas 29 Antidiabetic Agent Gramicidin D Bacillus brevis 11 Antibacterial Bacteria Agent Insulin Canine Pancreas Antidiabetic Agent Insulin A 21 Insulin B 30 Kallidin Human Plasma 10 Vasodilator Luteinizing Bovine 10 Hormone Hormone-Releasing Hypothalamus Stimulator Factor Melittin Bee Venom 26 Antirheumatic Agent Oxytocin Bovine Pituitary 9 Oxytocic Agent Gland Secretin Canine Intestine 27 Hormone Sermorelin Human Pancreas 29 Hormone Stimulator Somatostatin Bovine 14 Hormone Hypothalamus Inhibitor Vasopressin Bovine Pituitary 9 Antidiuretic Gland Agent

[0003] TABLE 2 Bioactive peptides utilized in applied research Size In Amino Name Isolated From Acids Biological Activity Atrial Natriuretic Rat Atria 28 Natriuretic Agent Peptide Bombesin Frog Skin 14 Gastric Activator Conantokin G Snail Venom 17 Neurotransmitter Conotoxin GI Snail Venom 13 Neuromuscular Inhibitor Defensin HNP-1 Human 30 Antimicrobial Agent Neutrophils Delta Sleep- Rabbit Brain 9 Neurological Affector Inducing Peptide Dermaseptin Frog Skin 34 Antimicrobial Agent Dynorphin Porcine Brain 17 Neurotransmitter EETI II Ecballium 29 Protease Inhibitor elaterium seeds Endorphin Human Brain 30 Neurotransmitter Enkephalin Human Brain 5 Neurotransmitter Histatin 5 Human Saliva 24 Antibacterial Agent Mastoparan Vespid Wasps 14 Mast Cell Degranulator Magainin I Frog Skin 23 Antimicrobial Agent Melanocyte Porcine 13 Hormone Stimulator Stimulating Pituitary Hormone Gland Motilin Canine 22 Gastric Activator Intestine Neurotensin Bovine Brain 13 Neurotransmitter Physalaemin Frog Skin 11 Hypotensive Agent Substance P Horse 11 Vasodilator Intestine Vasoactive Porcine 28 Hormone Intestinal Intestine Peptide

[0004] Where the mode of action of these peptides has been determined, it has been found to be due to the interaction of the bioactive peptide with a specific protein target. In most of the cases, the bioactive peptide acts by binding to and inactivating its protein target with extremely high specificities. Binding constants of these peptides for their protein targets typically have been determined to be in the nanomolar (nM, 10⁻⁹ M) range with binding constants as high as 10⁻¹² M (picomolar range) having been reported. Table 3 shows target proteins inactivated by several different bioactive peptides as well as the binding constants associated with binding thereto. TABLE 3 Binding constants of bioactive peptides Size in Bioactive Amino Peptide Acids Inhibited Protein Binding Constant α-Conotoxin GIA 15 Nicotinic 1.0 × 10⁻⁹ M Acetylcholine Receptor EETI II 29 Trypsin 1.0 × 10⁻¹² M H2(7-15) 8 HSV 3.6 × 10⁻⁵ M Ribonucleotide Reductase Histatin 5 24 Bacteroides 5.5 × 10⁻⁸ M gingivalis Protease Melittin 26 Calmodulin 3.0 × 10⁻⁹ M Myotoxin (29-42) 14 ATPase 1.9 × 10⁻⁵ M Neurotensin 13 Ni Regulatory 5.6 × 10⁻¹¹ M Protein Pituitary Adenylate 38 Calmodulin 1.5 × 10⁻⁸ M Cyclase Activating Polypeptide PKI (5-24) 20 cAMP-Dependent 2.3 × 10⁻⁹ M Protein Kinase SCP (153-180) 27 Calpain 3.0 × 10⁻⁸ M Secretin 27 HSR G Protein 3.2 × 10⁻⁹ M Vasoactive 28 GPRN1 G Protein 2.5 × 10⁻⁹ M Intestinal Peptide

[0005] Recently, there has been an increasing interest in employing synthetically derived bioactive peptides as novel pharmaceutical agents due to the impressive ability of the naturally occurring peptides to bind to and inhibit specific protein targets. Synthetically derived peptides could be useful in the development of new antibacterial, antiviral, and anticancer agents. Examples of synthetically derived antibacterial or antiviral peptide agents would be those capable of binding to and preventing bacterial or viral surface proteins from interacting with their host cell receptors, or preventing the action of specific toxin or protease proteins. Examples of anticancer agents would include synthetically derived peptides that could bind to and prevent the action of specific oncogenic proteins.

[0006] To date, novel bioactive peptides have been engineered through the use of two different in vitro approaches. The first approach produces candidate peptides by chemically synthesizing a randomized library of 6-10 amino acid peptides (J. Eichler et al., Med. Res. Rev. 15:481-496 (1995); K. Lam, Anticancer Drug Des. 12:145-167 (1996); M. Lebl et al., Methods Enzymol. 289:336-392 (1997)). In the second approach, candidate peptides are synthesized by cloning a randomized oligonucleotide library into a Ff filamentous phage gene, which allows peptides that are much larger in size to be expressed on the surface of the bacteriophage (H. Lowman, Ann. Rev. Biophys. Biomol. Struct. 26:401-424 (1997); G. Smith et al., et al. Meth. Enz. 217:228-257 (1993)). To date, randomized peptide libraries up to 38 amino acids in length have been made, and longer peptides are likely achievable using this system. The peptide libraries that are produced using either of these strategies are then typically mixed with a preselected matrix-bound protein target. Peptides that bind are eluted, and their sequences are determined. From this information new peptides are synthesized and their inhibitory properties are determined. This is a tedious process that only screens for one biological activity at a time.

[0007] Although these in vitro approaches show promise, the use of synthetically derived peptides has not yet become a mainstay in the pharmaceutical industry. The primary obstacle remaining is that of peptide instability within the biological system of interest as evidenced by the unwanted degradation of potential peptide drugs by proteases and/or peptidases in the host cells. There are three major classes of peptidases which can degrade larger peptides: amino and carboxy exopeptidases which act at either the amino or the carboxy terminal end of the peptide, respectively, and endopeptidases which act on an internal portion of the peptide. Aminopeptidases, carboxypeptidases, and endopeptidases have been identified in both prokaryotic and eukaryotic cells. Many of those that have been extensively characterized were found to function similarly in both cell types. Interestingly, in both prokaryotic and eukaryotic systems, many more aminopeptidases than carboxypeptidases have been identified to date.

[0008] Approaches used to address the problem of peptide degradation have included the use of D-amino acids or modified amino acids as opposed to the naturally occurring L-amino acids (e.g., J. Eichler et al., Med Res Rev. 15:481-496 (1995); L. Sanders, Eur. J. Drug Metabol. Pharmacokinetics 15: 95-102 (1990)), the use of cyclized peptides (e.g., R. Egleton, et al., Peptides 18:1431-1439 (1997)), and the development of enhanced delivery systems that prevent degradation of a peptide before it reaches its target in a patient (e.g., L. Wearley, Crit. Rev. Ther. Drug Carrier Syst. 8: 331-394 (1991); L. Sanders, Eur. J. Drug Metabol. Pharmacokinetics 15: 95-102 (1990)). Although these approaches for stabilizing peptides and thereby preventing their unwanted degradation in the biosystem of choice (e.g., a patient) are promising, there remains no way to routinely and reliably stabilize peptide drugs and drug candidates. Moreover, many of the existing stabilization and delivery methods cannot be directly utilized in the screening and development of novel useful bioactive peptides. A biological approach that would serve as both a method of stabilizing peptides and a method for identifying novel bioactive peptides would represent a much needed advance in the field of peptide drug development.

SUMMARY OF THE INVENTION

[0009] The present invention provides an intracellular screening method for identifying novel bioactive peptides. A host cell is transformed with an expression vector comprising a tightly regulable control region operably linked to a nucleic acid sequence encoding a peptide. The transformed host cell is first grown under conditions that repress expression of the peptide and then, subsequently, expression of the peptide is induced. Phenotypic changes in the host cell upon expression of the peptide are indicative of bioactivity, and are evaluated. If, for example, expression of the peptide is accompanied by inhibition of host cell growth, the expressed peptide constitutes a bioactive peptide, in that it functions as an inhibitory peptide.

[0010] Intracellular identification of bioactive peptides can be advantageously carried out in a pathogenic microbial host cell. Bioactive peptides having antimicrobial activity are readily identified in a microbial host cell system. Further, the method can be carried out in a host cell that has not been modified to reduce or eliminate the expression of naturally expressed proteases or peptidases. When carried out in a host cell comprising proteases and peptides, the selection process of the invention is biased in favor of bioactive peptides that are protease- and peptidase-resistant.

[0011] The tightly regulable control region of the expression vector used to transform the host cell according to the invention is preferably derived from the wild-type Escherichia coli lac operon, and the transformed host cell preferably comprises an amount of Lac repressor protein effective to repress expression of the peptide during host cell growth under repressed conditions. To insure a sufficient amount of Lac repressor protein, the host cell can be transformed with a second vector the overproduces Lac repressor protein.

[0012] Optionally, the expression vector used to transform the host cell can be genetically engineered to encode a stabilized peptide that is resistant to peptidases and proteases. For example, the coding sequence can be designed to encode a stabilizing group at either or both of the peptide N-terminus or C-terminus. As another example, the coding sequence can be designed to encode a stablizing motif such as an α-helix motif or an opposite charge ending motif, as described below. The presence of a stabilizing group at a peptide terminus or a stabilizing motif can slow down the rate of intracellular degradation of the peptide.

[0013] The invention further provides a bioactive peptide having a first stabilizing group comprising the N-terminus and a second stabilizing group comprising the C-terminus. Preferably, the first stabilizing group is selected from the group consisting of a small stable protein, Pro-, Pro-Pro-, Xaa-Pro- and Xaa-Pro-Pro-; and the second stabilizing group is selected from the group consisting of a small stable protein, -Pro, -Pro-Pro, -Pro-Xaa and -Pro-Pro-Xaa. Suitable small stable proteins include Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, and glutathione reductase. In addition, the invention provides a bioactive peptide stabilized by an opposite charge ending. motif, as described below. The bioactive peptide is preferably an antimicrobial peptide or a therapeutic peptide drug.

[0014] Also provided by the invention is a polypeptide that can be cleaved to yield a bioactive peptide having a stabilizing group at either or both of its N- and C-termini. The cleavable polypeptide accordingly comprises a chemical or enzymatic cleavage site either immediately preceding the N-terminus of the bioactive peptide or immediately following the C-terminus of the bioactive peptide.

[0015] The invention further provides a fusion protein comprising a four-helix bundle protein, preferably the Rop protein, and a polypeptide. The four-helix bundle protein is positioned at either the N-terminus or the C-terminus of the fusion protein, and accordingly can be fused to either the N-terminus or the C-terminus of the polypeptide.

[0016] The present invention also provides a method for using an antimicrobial peptide. An antimicrobial peptide is stabilized by linking a first stabilizing group to the N-terminus of an antimicrobial peptide, and, optionally, a second stabilizing group to the C-terminus of the antimicrobial peptide. Alternatively, the antimicrobial peptide is stabilized by flanking the peptide sequence with an opposite charge ending motif, as described below. The resulting stabilized antimicrobial peptide is brought into contact with a microbe, preferably a pathogenic microbe, for example to inhibit the growth or toxicity of the microbe.

[0017] The invention also provides a method for treating a patient having a condition treatable with a peptide drug, comprising administering to the patient a stabilized peptide drug having at least one of a first stabilizing group comprising the N-terminus of the stabilized peptide drug and a second stabilizing group comprising the C-terminus of the stabilized peptide drug. Optionally, prior to administration of the stabilized peptide drug, the first stabilizing group is covalently linked to the N-terminus of a peptide drug, and the second stabilizing group is covalently linked to the C-terminus of the peptide drug to yield the stabilized peptide drug. Alternatively, the method comprises administering to the patient a peptide drug that has been stabilized by flanking the peptide sequence with an opposite charge ending motif, as described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 shows the control region (SEQ ID NO: 1) of the wild-type lac operon from the auxiliary operator O3 through the translational start of the lacZ gene. DNA binding sites include the operators O3 and O1 (both underlined), CAP (boxed), the −35 site (boxed), and the −10 site (boxed), while important RNA and protein sites include the LacI translation stop site (TGA), the +1 lacZ transcription start site, the Shine Dalgarno. (SD) ribosome binding site for lacZ, and the LacZ translation start site (ATG).

[0019]FIG. 2 is a map of plasmid pLAC11. The unique restriction sites and the base pair at which they cut are indicated. Other sites of interest are also shown, including Tet (98-1288), Rop (1931-2122), ori (2551-3138), Amp (3309-4169), and lacPO (4424-4536).

[0020]FIG. 3 is a map of plasmid pLAC22. The unique restriction sites and the base pair at which they cut are indicated. Other sites of interest are also shown, including Tet (98-1288), Rop (1927-2118), ori (2547-3134), Amp (3305-4165), lacIq (4452-5536), and lacPO (5529-5641).

[0021]FIG. 4 is a map of plasmid pLAC33. The unique restriction sites and the base pair at which they cut are indicated. Other sites of interest are also shown, including Tet (98-1288), ori (1746-2333), Amp (2504-3364), and lacPO (3619-3731).

[0022]FIG. 5 shows the response of the pLAC11-lacZ construct (open circles) to varying amounts of isopropyl β-D-thiogalactoside (IPTG). A filled square indicates the β-galactosidase activity that was obtained when MG1655 or CSH27 cells were grown in rich media induced with 1 mM IPTG, while a filled diamond indicates the β-galactosidase activity that was obtained when MG1655 or CSH27 cells were grown in M9 minimal lactose media.

[0023]FIG. 6 shows growth curves depicting the inhibitory effects of a two day inhibitor (pPep12) versus a one day inhibitor (pPep1). Data points for the control, pLAC11, for pPep1, and for pPep12, are indicated by squares, circles, and triangles, respectively.

[0024]FIG. 7 is a map of the p-Rop(C) fusion vector. The unique restriction sites and the base pair at which they cut are indicated. Other sites of interest are also shown, including Rop (7-198), ori (627-1214), Amp (2245-1385), lacPO (2500-2612).

[0025]FIG. 8 is a map of the p(N)Rop-fusion vector. The unique restriction sites and the base pair at which they cut are indicated. Other sites of interest are also shown; Rop (7-204), ori (266-853), Amp (1024-1884), lacPO (2139-2251).

[0026]FIG. 9 illustrates a peptide (SEQ ID NO:2) having the opposite charge ending motif, wherein the amino and carboxy termini of the peptide are stabilized by the interactions of the opposite charge ending amino acids.

DETAILED DESCRIPTION

[0027] The present invention represents a significant advance in the art of peptide drug development by allowing concurrent screening for peptide bioactivity and stability. Randomized recombinant peptides are screened for bioactivity in a tightly regulated inducible expression system, preferably derived from the wild-type lac operon, that permits essentially complete repression of peptide expression in the host cell. Subsequent induction of peptide expression can then be used to identify peptides that inhibit host cell growth or possess other bioactivities.

[0028] Intracellular screening of randomized peptides has many advantages over existing methods. Bioactivity is readily apparent, many diverse bioactivities can be screened for simultaneously, very large numbers of peptides can be screened using easily generated peptide libraries, and the host cell, if desired, can be genetically manipulated to elucidate an affected protein target. Advantageously, randomized peptides can be screened in a host cell that is identical to or closely resembles the eventual target cell for antimicrobial applications. An additional and very important feature of this system is that selection is naturally biased in favor of peptides that are stable in an intracellular environment; i.e., that are resistant to proteases and peptidases. Fortuitously, bacterial peptidases are very similar to eukaryotic peptidases. Peptides that are stable in a bacterial host are thus likely to be stable in a eukaryotic cell as well, allowing bacterial cells to be used in initial screens to identify drugs that may eventually prove useful as human or animal therapeutics.

[0029] The invention is directed to the identification and use of bioactive peptides. A bioactive peptide is a peptide having a biological activity. The term “bioactivity” as used herein includes, but is not limited to, any type of interaction with another biomolecule, such as a protein, glycoprotein, carbohydrate, for example an oligosaccharide or polysaccharide, nucleotide, polynucleotide, fatty acid, hormone, enzyme, cofactor or the like, whether the interactions involve covalent or noncovalent binding. Bioactivity further includes interactions of any type with other cellular components or constituents including salts, ions, metals, nutrients, foreign or exogenous agents present in a cell such as viruses, phage and the like, for example binding, sequestration or transport-related interactions. Bioactivity of a peptide can be detected, for example, by observing phenotypic effects in a host cell in which it is expressed, or by performing an in vitro assay for a particular bioactivity, such as affinity binding to a target molecule, alteration of an enzymatic activity, or the like. Examples of bioactive peptides include antimicrobial peptides and peptide drugs. Antimicrobial peptides are peptides that adversely affect a microbe such as a bacterium, virus, protozoan, or the like. Antimicrobial peptides include, for example, inhibitory peptides that slow the growth of a microbe, microbiocidal peptides that are effective to kill a microbe (e.g., bacteriocidal and virocidal peptide drugs, sterilants, and disinfectants), and peptides effective to interfere with-microbial reproduction, host toxicity, or the like. Peptide drugs for therapeutic use in humans or other animals include, for example, antimicrobial peptides that are not prohibitively toxic to the patient, peptides designed to elicit, speed up, slow down, or prevent various metabolic processes in the host such as insulin, oxytocin, calcitonin, gastrin, somatostatin, anticancer peptides, and the like.

[0030] The term “peptide” as used herein refers to a plurality of amino acids joined together in a linear chain via peptide bonds. Accordingly, the term “peptide” as used herein includes a dipeptide, tripeptide, oligopeptide and polypeptide. A dipeptide contains two amino acids; a tripeptide contains three amino acids; and the term oligopeptide is typically used to describe peptides having between 2 and about 50 or more amino acids. Peptides larger than about 50 are often referred to polypeptides or proteins. For purposes of the present invention, a “peptide” is not limited to any particular number of amino acids. Preferably, however, the peptide contains about 2 to about 50 amino acids, more preferably about 5 to about 40 amino acids, most preferably about 5 to about 20 amino acids.

[0031] The library used to transform the host cell is formed by cloning a randomized, peptide-encoding oligonucleotide into a nucleic acid construct having a tightly regulable expression control region. An expression control region can be readily evaluated to determine whether it is “tightly regulable,” as the term is used herein, by bioassay in a host cell engineered to contain a mutant nonfunctional gene “X.” Transforming the engineered host cell with an expression vector containing a tightly regulable expression control region operably linked to a cloned wild-type gene “X” will preserve the phenotype of the engineered host cell under repressed conditions. Under induced conditions, however, the expression vector containing the tightly regulable expression control region that is operably linked to the cloned wild-type gene “X” will complement the mutant nonfunctional gene X to yield the wild-type phenotype. In other words, a host cell containing a null mutation which is transformed with a tightly regulable expression vector capable of expressing the chromosomally inactivated gene will exhibit the null phenotype under repressed conditions; but when expression is induced the cell will exhibit a phenotype indistinguishable from the wild-type cell. It should be understood that the expression control region in the tightly regulable expression vector of the present invention can be readily modified to produce higher levels of an encoded biopeptide, if desired (see, e.g., Example I, below). Such modification may unavoidably introduce some “leakiness” into expression control, resulting in a low level of peptide expression under repressed conditions.

[0032] In a preferred embodiment, the expression control region of the inducible expression vector is derived from the wild-type E. coli lac promoter/operator region. In a particularly preferred form, the expression vector contains a regulatory region that includes the auxiliary operator O3, the CAP binding region, the −35 promoter site, the −10 promoter site, the operator O1, the Shine-Dalgarno sequence for lacZ, and a spacer region between the end of the Shine-Dalgarno sequence and the ATG start of the lacZ coding sequence (see FIG. 1).

[0033] It is to be understood that variations in the wild-type nucleic acid sequence of the lac promoter/operator region can be tolerated in the expression control region of the preferred expression vector and are encompassed by the invention, provided that the expression control region remains tightly regulable as defined herein. For example, the −10 site of the wild-type lac operon (TATGTT) is weak compared to the bacterial consensus −10 site sequence TATAAT, sharing four out of six positions. It is contemplated that other comparably weak promoters are equally effective at the −10 site in the expression control region; a strong promoter is to be avoided in order to insure complete repression in the uninduced state. With respect to the −35 region, the sequence of the wild-type lac operon, TTTACA, is one base removed from the consensus −35 sequence TTGACA. It is contemplated that a tightly regulable lac operon-derived expression control region could be constructed using a weaker −35 sequence (i.e., one having less identity with the consensus −35 sequence) and a wild-type −10 sequence (TATAAT), yielding a weak promoter that needs the assistance of the CAP activator protein. Similarly, it is to be understood that the nucleic acid sequence of the CAP binding region can be altered as long as the CAP protein binds to it with essentially the same affinity. The spacer region between the end of the Shine-Dalgarno sequence and the ATG start of the lacZ coding sequence is typically between about 5 and about 10 nucleotides in length, preferably about 5 to about 8 nucleotides in length, more preferably about 7-9 nucleotides in length. The most preferred composition and length of the spacer region depends on the composition and length of Shine-Dalgarno sequence with which it is operably linked as well as the translation start codon employed (i.e., AUG, GUG, or UUG), and can be determined accordingly by one of skill in the art. Preferably the nucleotide composition of the spacer region is “AT rich”; that is, it contains more A's and T's than it does G's and C's.

[0034] In a preferred embodiment of the method of the invention, the expression vector has the identifying characteristics of pLAC11 (ATCC No. 207108). More preferably, the expression vector is pLAC11 (ATCC No. 207108).

[0035] As used in the present invention, the term “vector” is to be broadly interpreted as including a plasmid, including an episome, a viral vector, a cosmid, or the like. A vector can be circular or linear, single-stranded or double-stranded, and can comprise RNA, DNA, or modifications and combinations thereof. Selection of a vector or plasmid backbone depends upon a variety of characteristics desired in the resulting construct, such as selection marker(s), plasmid copy number, and the like. A nucleic acid sequence is “operably linked” to an expression control sequence in the regulatory region of a vector, such as a promoter, when the expression control sequence controls or regulates the transcription and/or the translation of that nucleic acid sequence. A nucleic acid that is “operably linked” to an expression control sequence includes, for example, an appropriate start signal (e.g., ATG) at the beginning of the nucleic acid sequence to be expressed and a reading frame that permits expression of the nucleic acid sequence under control of the expression control sequence to yield production of the encoded peptide. The regulatory region of the expression vector optionally includes a termination sequence, such as a codon for which there is no corresponding aminoacetyl-tRNA, thus ending peptide synthesis. Typically, when the ribosome reaches a termination sequence or codon during translation of the mRNA, the polypeptide is released and the ribosome-mRNA-tRNA complex dissociates.

[0036] An expression vector optionally includes one or more selection or marker sequences, which typically encode an enzyme capable of inactivating a compound in the growth medium. The inclusion of a marker sequence can, for example, render the host cell resistant to an antibiotic, or it can confer a compound-specific metabolic advantage on the host cell. Cells can be transformed with the expression vector using any convenient method known in the art, including chemical transformation, e.g., whereby cells are made competent by treatment with reagents such as CaCl₂; electroporation and other electrical techniques; microinjection and the like.

[0037] In embodiments of the method that make use of a tightly regulable expression system derived from the lac operon, the host cell is or has been genetically engineered or otherwise altered to contain a source of Lac repressor protein in excess of the amount produced in wild-type E. coli. A host cell that contains an excess source of Lac repressor protein is one that expresses an amount of Lac repressor protein sufficient to repress expression of the peptide under repressed conditions, i.e., in the absence of an inducing agent, such as isopropyl β-D-thiogalactoside (IPTG). Preferably, expression of Lac repressor protein is constitutive; For example, the host cell can be transformed with a second vector comprising a gene encoding Lac repressor protein, preferably lacI, more preferably lacI^(q), to provide an excess source of Lac repressor protein in trans, i.e., extraneous to the tightly regulable expression vector. An episome can also serve as a trans source of Lac repressor. Another option for providing a trans source of Lac repressor protein is the host chromosome itself, which can be genetically engineered to express excess Lac repressor protein Alternatively, a gene encoding Lac repressor protein can be included on the tightly regulable expression vector that contains the peptide-encoding oligonucleotide so that Lac repressor protein is provided in cis. The gene encoding the Lac repressor protein is preferably under the control of a constitutive promoter.

[0038] The invention is not intended to be limited in any way by the type of host cell used for screening. The host cell can be a prokaryotic or a eukaryotic cell. Preferred mammalian cells include human cells, of any tissue type, and can include cancer cells or hybridomas, without limitation. Preferred bacterial host cells include gram negative bacteria, such as E. coli and various Salmonella spp., and gram positive bacteria, such as bacteria from the genera Staphylococcus, Streptococcus and Enterococcus. Protozoan cells are also suitable host cells. In clear contrast to conventional recombinant protein expression systems, it is preferable that the host cell contains proteases and/or peptidases, since the selection will, as a result, be advantageously biased in favor of peptides that are protease- and peptidase-resistant. More preferably, the host cell has not been modified, genetically or otherwise, to reduce or eliminate the expression of any naturally expressed proteases or peptidases. The host cell can be selected with a particular purpose in mind. For example, if it is desired to obtain peptide drugs specific to inhibit Staphylococcus, peptides can be advantageously expressed and screened in Staphylococcus.

[0039] There is, accordingly, tremendous potential for the application of this technology in the development of new antibacterial peptides useful to treat various pathogenic bacteria. Of particular interest are pathogenic Staphylococci, Streptococci, and Enterococci, which are the primary causes of nosocomial infections. Many of these strains are becoming increasingly drug-resistant at an alarming rate. The technology of the present invention can be practiced in a pathogenic host cell to isolate inhibitor peptides that specifically target the pathogenic strain of choice. Inhibitory peptides identified using pathogenic microbial host cells in accordance with the invention may have direct therapeutic utility; based on what is known about peptide import, it is very likely that small peptides are rapidly taken up by Staphylococci, Streptococci, and Enterococci. Once internalized, the inhibitory peptides identified according to the invention would be expected to inhibit the growth of the bacteria in question. It is therefore contemplated that novel inhibitor peptides so identified can be used in medical treatments and therapies directed against microbial infection. It is further contemplated that these novel inhibitor peptides can be used, in turn, to identify additional novel antibacterial peptides using a synthetic approach. The coding sequence of the inhibitory peptides is determined, and peptides are then chemically synthesized and tested in the host cell for their inhibitory properties.

[0040] Novel inhibitor peptides identified in a pathogenic microbial host cell according to the invention can also be used to elucidate potential new drug targets. The protein target that the inhibitor peptide inactivated is identified using reverse genetics by isolating mutants that are no longer inhibited by the peptide. These mutants are then mapped in order to precisely determine the protein target that is inhibited. New antibacterial drugs can then be developed using various known or yet to be discovered pharmaceutical strategies.

[0041] Following transformation of the host cell, the transformed host cell is initially grown under conditions that repress expression of the peptide. Expression of the peptide is then induced. For example, when a lac promoter/operator system is used for expression, IPTG is added to the culture medium. A determination is subsequently made as to whether the peptide is inhibitory to host cell growth, wherein inhibition of host cell growth under induced but not repressed conditions is indicative of the expression of a bioactive peptide.

[0042] Notably, the bioactive peptides identified according to the method of the invention are, by reason of the method itself, stable in the intracellular environment of the host cell. The method of the invention thus preferably identifies bioactive peptides that are resistant to proteases and peptidases.

[0043] Resistance to proteases and peptidases can be evaluated by measuring peptide degradation when in contact with appropriate cell extracts or purified peptidases and/or proteases, employing methods well-known in the art. A protease- or peptidase-resistant peptide is evidenced by a longer half-life in the presence of proteases or peptidases compared to a control peptide.

[0044] Randomized peptides used in the screening method of the invention can be optionally engineered according to the method of the invention in a biased synthesis to increase their stability by making one or both of the N-terminal or C-terminal ends more resistant to proteases and peptidases, and/or by engineering into the peptides a stabilizing motif.

[0045] In one embodiment of the screening method of the invention, the putative bioactive peptide is stabilized by adding a stabilizing group to the N-terminus, the C-terminus, or to both termini. To this end, the nucleic acid sequence that encodes the randomized peptide in the expression vector or the expression vector itself is preferably modified to encode a first stabilizing group comprising the N-terminus of the peptide, and a second stabilizing group comprising the C-terminus of the peptide.

[0046] The stabilizing group can be a stable protein, preferably a small stable protein such as thioredoxin, glutathione sulfotransferase, maltose binding protein, glutathione reductase, or a four-helix bundle protein such as Rop protein, although no specific size limitation on the protein anchor is intended. Proteins suitable for use as a stabilizing group can be either naturally occurring or non-naturally occurring. They can be isolated from an endogenous source, chemically or enzymatically synthesized, or produced using recombinant DNA technology. Proteins that are particularly well-suited for use as a stabilizing group are those that are relatively short in length and form very stable structures in solution. Proteins having molecular weights of less than about 50 kD are preferred for use as a stabilizing group; more preferably the molecular weight of the small stable protein is less than about 25 kD, most preferably less than about 12 kD. For example, E. coli thioredoxin has a molecular weight about 11.7 kD; E. coli glutathione sulfotransferase has a molecular weight of about 22.9 kD, and Rop from the ColE1 replicon has a molecular weight of about 7.2 kD); and maltose binding protein (without its signal sequence) is about 40.7 kD. The small size of the Rop protein makes it especially useful as a stabilizing group, fusion partner, or peptide anchor, in that it is less likely than larger proteins to interfere with the accessibility of the linked peptide, thus preserving its bioactivity. Rop's highly ordered anti-parallel four-helix bundle topology (after dimerization) and slow unfolding kinetics ( see, e.g., Betz et al, Biochemistry 36, 2450-2458 (1997)) also contribute to its usefulness as a peptide anchor according to the invention. Other proteins with similar folding kinetics and/or thermodynamic stability (e.g., Rop has a midpoint temperature of denaturation, T_(m), of about 71° C., Steif et al., Biochemistry 32, 3867-3876 (1993)) are also preferred peptide anchors. Peptides or proteins having highly stable tertiary motifs, such as a four-helix bundle topology, are particularly preferred.

[0047] Alternatively, the stabilizing group can constitute one or more prolines (Pro). Preferably, a proline dipeptide (Pro-Pro) is used as a stabilizing group, however additional prolines may be included. The encoded proline(s) are typically naturally occurring amino acids, however if and to the extent a proline derivative, for example a hydroxyproline or a methyl- or ethyl-proline derivative, can be encoded or otherwise incorporated into the peptide, those proline derivatives are also useful as stabilizing groups.

[0048] At the N-terminus of the peptide, the stabilizing group can alternatively include an oligopeptide having the sequence Xaa-Pro_(m)-, wherein Xaa is any amino acid, and m is greater than 0. Preferably, m is about 1 to about 5; preferably m=2 or 3, more preferably, m=2. Likewise, at the C-terminus of the peptide, the stabilizing group can alternatively include an oligopeptide having the sequence -Pro_(m)-Xaa, wherein Xaa is any amino acid, and m is greater than 0. Preferably, n is about 1 to about 5; preferably n=2 or 3, more preferably, m=2. In a particularly preferred embodiment of the method of the invention, the nucleic acid sequence that encodes the randomized peptide in the expression vector is modified to encode each of a first stabilizing group comprising the N-terminus of the peptide, the first stabilizing group being selected from the group consisting of small stable protein, Pro-, Pro-Pro-, Xaa-Pro- and Xaa-Pro-Pro-, and a second stabilizing group comprising the C-terminus of the peptide, the second stabilizing group being selected from the group consisting of a small stable protein, -Pro, -Pro-Pro, Pro-Xaa and Pro-Pro-Xaa. The resulting peptide has enhanced stability in the intracellular environment relative to a peptide lacking the terminal stabilizing groups.

[0049] In another preferred embodiment of the screening method of the invention, the expression vector encodes a four-helix bundle protein fused, at either the C-terminus or the N-terminus, to the randomized peptide. Preferably, the four-helix bundle protein is E. coli Rop protein or a homolog thereof. The non-fused terminus of the randomized peptide can, but need not, comprise a stabilizing group. The resulting fusion protein is predicted to be more stable than the randomized peptide itself in the host intracellular environment. Where the four-helix bundle protein is fused to the N-terminus, the randomized peptide can optionally be further stabilized by engineering one or more prolines, with or without a following undefined amino acid (e.g., -Pro, -Pro-Pro, -Pro-Xaa, -Pro-Pro-Xaa, etc.) at the C-terminus of the peptide sequence; likewise, when the four-helix bundle protein is fused to the C-terminus, the randomized peptide can be further stabilized by engineering one or more prolines, with or without a preceding undefined amino acid (e.g., Pro-, Pro-Pro-, Xaa-Pro-, Xaa-Pro-Pro-, etc.) at the N-terminus of the peptide sequence.

[0050] In yet another embodiment of the screening method of the invention, the putative bioactive peptide is stabilized by engineering into the peptide a stabilizing motif such as an α-helix motif or an opposite charge ending motif. Chemical synthesis of an oligonucleotide according to the scheme [(CAG)A(TCAG)] yields an oligonucleotide encoding a peptide consisting of a random mixture of the hydrophilic amino acids His, Gin, Asn, Lys, Asp, and Glu (see Table 14). Except for Asp, these amino acids are most often associated with α-helical secondary structural motifs; the resulting oligonucleotides are thus biased in favor of oligonucleotides that encode peptides that are likely to form α-helices in solution. Alternatively, the putative bioactive peptide is stabilized by flanking a randomized region with a region of uniform charge (e.g., positive charge) on one end and a region of opposite charge (e.g., negative) on the other end, to form an opposite charge ending motif. To this end, the nucleic acid sequence that encodes the randomized peptide in the expression vector or the expression vector itself is preferably modified to encode a plurality of sequential uniformly charged amino acids comprising the N-terminus of the peptide, and a plurality of sequential oppositely charged amino acids comprising the C-terminus of the peptide. The positive charges are supplied by a plurality of positively charged amino acids consisting of lysine, histidine, arginine or a combination thereof; and the negative charges are supplied by a plurality of negatively charged amino acids consisting of aspartate, glutamate or a combination thereof. It is expected that such a peptide will be stabilized by the ionic interaction of the two oppositely charges ends. Preferably, the putative bioactive peptide contains at least three charged amino acids at each end. More preferably, it contains at least four charged amino acids at each end. In a particularly preferred embodiment, the larger acidic amino acid glutamate is paired with the smaller basic amino acid lysine, and the smaller acidic amino acid aspartate is paired with the larger basic amino acid arginine.

[0051] It is to be understood that novel bioactive peptides identified using the method for identification of bioactive peptides described herein are also included in the present invention.

[0052] The present invention further provides a bioactive peptide containing one or more structural features or motifs selected to enhance the stability of the bioactive peptide in an intracellular environment. During development and testing of the intracellular screening method of the present invention, it was surprisingly discovered that several bioactive peptides identified from the randomized peptide library shared particular structural features. For example, a disproportionately high number of bioactive peptides identified using the intracellular screening method contained one or more proline residues at or near a peptide terminus. A disproportionate number also contained sequences predicted, using structure prediction algorithms well-known in the art, to form secondary structures such as α helices or β sheets; or a hydrophobic membrane spanning domain. Bioactive fusion proteins comprising the randomized peptide sequence fused to the Rop protein, due to a deletion event in the expression vector, were also identified.

[0053] Accordingly, the invention provides a bioactive peptide having a stabilizing group at its N-terminus, its C-terminus, or at both termini. In a bioactive peptide stabilized at only one terminus (i.e., at either the N- or the C-terminus) the stabilizing group is preferably either a four-helix bundle protein, such as Rop protein, a proline (Pro), or a proline dipeptide (Pro-Pro). It should be understood that in any synthetic peptide having a stabilizing group that includes one or more prolines according to the present invention, the proline is preferably a naturally occurring amino acid; alternatively, it can be a synthetic derivative of proline, for example a hydroxyproline or a methyl- or ethyl- proline derivative. Accordingly, where the abbreviation “Pro” is used herein in connection with a stabilizing group that is part of a synthetic peptide, it is meant to include proline derivatives in addition to a naturally occurring proline.

[0054] A peptide stabilized at both termini includes a first stabilizing group comprising the N-terminus, and a second stabilizing group stabilizing the C-terminus, where the first and second stabilizing groups are as defined previously in connection with the method for identifying bioactive peptides. The stabilizing group is covalently attached to the peptide. The bioactive peptide of the invention includes a bioactive peptide that has been detectably labeled, derivatized, or modified in any manner desired prior to use, provided it contains one or more terminal stabilizing groups as provided herein. In one preferred embodiment of the bioactive peptide of the invention, the first stabilizing group, comprising the N-terminus, is Xaa-Pro-Pro-, Xaa-Pro-, Pro- or Pro-Pro-; and second stabilizing group, comprising the C-terminus, is Pro-Pro-Xaa, -Pro-Xaa, -Pro or -Pro-Pro; preferably -Pro-Pro. In another preferred embodiment, the first (N-terminal) stabilizing group is a small stable protein, preferably a four-helix bundle protein such as Rop protein; and the second (C-terminal) stabilizing group is Pro-Pro-Xaa, -Pro-Xaa, -Pro or -Pro-Pro; preferably -Pro-Pro. In yet another preferred embodiment, the second (C-terminal) stabilizing group is a small stable protein, preferably a four-helix bundle protein such as Rop protein, and the first (N-terminal) stabilizing group is Pro-, Pro-Pro-, Xaa-Pro- or Xaa-Pro-Pro-.

[0055] The invention further provides a peptide stabilized by flanking the amino acid sequence of a bioactive peptide with an opposite charge ending motif, as described herein. Preferably, the resulting stabilized peptide retains at least a portion of the biological activity of the bioactive protein. The stabilized peptide includes a peptide that has been detectably labeled, derivatized, or modified in any manner desired prior to use.

[0056] It should be understood that any bioactive peptide, without limitation, can be stabilized according to the invention by attaching a stabilizing group to either or both of the N- and C-termini, or by attaching oppositely charged groups to the N- and C-termini to form an opposite charge ending motif. Included in the present invention are any and various antimicrobial peptides, inhibitory peptides, therapeutic peptide drugs, and the like, as, for example and without limitation, those listed in Tables 1 and 2, that have been modified at one or both peptide termini to include a stabilizing group, for example a four-helix bundle protein such as Rop protein, proline (Pro-), a proline-proline dipeptide-(Pro-Pro-), an Xaa-Pro- dipeptide, or an Xaa-Pro-Pro-tripeptide at the N-terminus, and/or a four-helix bundle protein such as Rop protein, proline (-Pro), or a proline-proline dipeptide (-Pro-Pro), a Pro-Xaa dipeptide, or a Pro-Pro-X tripeptide at the C-terminus; or that have been modified to contain an opposite charge ending motif according to the invention. In this aspect the invention is exemplified by peptides such as Pro-Pro-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Ile-Pro-Pro (SEQ ID NO: 3) and Glu-Asp-Glu-Asp-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Ile-Arg-Lys-Arg-Lys (SEQ ID NO: 4), wherein the middle nine amino acids (-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Ile-; SEQ ID NO: 5) constitute the sequence of angiotensin.

[0057] Modification of a bioactive peptide to yield a stabilized bioactive peptide according to the invention can be achieved by standard techniques well-known in the arts of genetics and peptide synthesis. For example, where the peptide is synthesized de novo, as in solid state peptide synthesis, one or more prolines can be added at the beginning and the end of the peptide chain during the synthetic reaction. In recombinant synthesis, for example as described in Example III herein, one or more codons encoding proline can be inserted into the peptide coding sequence at the beginning and/or the end of the sequence, as desired. Preferably, codons encoding N-terminal prolines are inserted after (i.e., 3′ to) the initiation site ATG (which encodes for methionine). Analogous techniques are used to synthesize bioactive peptides having an opposite charge ending motif. When a known bioactive peptide is modified to yield a stabilized bioactive peptide according to the invention, the unmodified peptide can conveniently be used as a control in a protease- or peptidase-resistance assay as described hereinabove to confirm, if desired, that the modified peptide exhibits increased stability.

[0058] The present invention also provides a cleavable polypeptide comprising a stabilized, bioactive peptide either immediately preceded by (i.e., adjacent to the N-terminus of the bioactive peptide) a cleavage site, or immediately followed by (i.e., adjacent to the C-terminus of the bioactive peptide) a cleavage site. Thus, a bioactive peptide as contemplated by the invention can be part of a cleavable polypeptide. The cleavable polypeptide is cleavable, either chemically, as with cyanogen bromide, or enzymatically, to yield the bioactive peptide. The resulting bioactive peptide either includes a first stabilizing group comprising its N-terminus and a second stabilizing group comprising its C-terminus, or it includes an opposite charge ending motif, both as described hereinabove. The cleavage site immediately precedes the N-terminal stabilizing group or immediately follows the C-terminal stabilizing group. In the case of a bioactive peptide having an opposite charge ending motif, the cleavage site immediately precedes the first charged region or immediately follows the second charged region. The cleavage site makes it possible to administer a bioactive peptide in a form that could allow intracellular targeting and/or activation.

[0059] Alternatively, a bioactive peptide of the invention can be fused to a noncleavable N-terminal or C-terminal targeting sequence wherein the targeting sequence allows target,ed delivery of the bioactive peptide, e.g., intracellular targeting or tissue-specific targeting of the bioactive peptide. In one embodiment of this aspect of the invention, the free terminus of the bioactive peptide comprises a stabilizing group as described hereinabove in connection with the screening method for identifying bioactive peptides, for example one or more prolines. The targeting sequence forming the other peptide terminus can, but need not, contain a small stable protein such as Rop or one or more prolines comprising its terminus, as long as the targeting function of the targeting sequence is preserved. In another embodiment of this aspect of the invention, the bioactive peptide comprises a charge ending motif as described hereinabove, wherein one charged region occupies the free terminus of the bioactive peptide, and the other charged region is disposed between the targeting sequence and the active sequence of the bioactive peptide.

[0060] The invention further includes a method for using an antimicrobial peptide that includes covalently linking a stabilizing group, as described hereinabove, to the N-terminus, the C-terminus, or to both termini, to yield a stabilized antimicrobial peptide, then contacting a microbe with the stabilized antimicrobial peptide. Alternatively, the stabilized antimicrobial peptide used in this aspect of the invention is made by covalently linking oppositely charged regions, as described hereinabove, to each end of the antimicrobial peptide to form an opposite charge ending motif. An antimicrobial peptide is to be broadly understood as including any bioactive peptide that adversely affects a microbe such as a bacterium, virus, protozoan, or the like, as described in more detail hereinabove. An example of an antimicrobial peptide is an inhibitory peptide that inhibits the growth of a microbe. When the antimicrobial peptide is covalently linked to a stabilizing group at only one peptide terminus, any of the stabilizing groups described hereinabove can be utilized. When the antimicrobial peptide is covalently linked to a stabilizing group at both peptide termini, the method includes covalently linking a first stabilizing group to the N-terminus of the antimicrobial peptide and a second stabilizing group to the C-terminus of the antimicrobial peptide, where the first and second stabilizing groups are as defined previously in connection with the method for identifying bioactive peptides. In a preferred embodiment of the method for using an antimicrobial peptide, one or more prolines, more preferably a proline-proline dipeptide, is attached to at least one, preferably both, termini of the antimicrobial peptide. Alternatively, or in addition, an Xaa-Pro- or an Xaa-Pro-Pro sequence can be attached to the N-terminus of a microbial peptide, and/or a Pro-Xaa or a Pro-Pro-Xaa sequence can be attached to the C-terminus, to yield a stabilized antimicrobial peptide.

[0061] The antimicrobial peptide thus modified in accordance with the invention has enhanced stability in the intracellular environment relative to an unmodified antimicrobial peptide. As noted earlier, the unmodified peptide can conveniently be used as a control in a protease- or peptidase-resistance assay as described hereinabove to confirm, if desired, that the modified peptide exhibits increased stability. Further, the antimicrobial activity of the antimicrobial peptide is preferably preserved or enhanced in the modified antimicrobial peptide; modifications that reduce or eliminate the antimicrobial activity of the antimicrobial peptide are easily detected and are to be avoided.

[0062] The invention further provides a method for inhibiting the growth of a microbe comprising contacting the microbe with a stabilized inhibitory peptide. In one embodiment of this aspect of the invention, the stabilized inhibitory peptide has a stabilizing group at the N-terminus, the C-terminus, or to both. Preferably, the inhibitory peptide has a first stabilizing group comprising the N-terminus of the inhibitory peptide, and a second stabilizing group comprising the C-terminus of the inhibitory peptide; the first and second stabilizing groups are as defined previously in connection with the method for identifying bioactive peptides. In another embodiment of this aspect of the invention, the inhibitory peptide is stabilized by the addition of oppositely charged regions to each end to form an opposite charge ending motif, as described hereinabove.

[0063] Also included in the present invention is a method for treating a patient having a condition treatable with a peptide drug, comprising administering to the patient a peptide drug that has been stabilized as described herein. Peptide drugs for use in therapeutic treatments are well-known (see Table 1). However, they are often easily degraded in biological systems, which affects their efficacy. In on embodiment of the present method, the patient is treated with a stabilized drug comprising the peptide drug of choice and a stabilizing group attached at either the N-terminus, the C-terminus of, or at both termini of the peptide drug. In another embodiment of the present method, the patient is treated with a stabilized drug comprising the peptide drug of choice and stabilized by attachment of oppositely charged regions to both termini of the peptide drug. Because the peptide drug is thereby stabilized against proteolytic degradation, greater amounts of the drug should reach the intended target in the patient.

[0064] In embodiments of the method involving administration of a peptide drug that is covalently linked to a stabilizing group at only one peptide terminus, the stabilizing group is preferably a four-helix bundle protein such as Rop protein, provided that attachment of the four-helix bundle protein to the peptide terminus preserves a sufficient amount of efficacy for the drug. It is to be nonetheless understood that group or groups used to stabilize the peptide drug are as defined hereinabove, without limitation. In embodiments involving administration of a peptide drug covalently linked to a stabilizing group at both peptide termini, the peptide drug includes a first stabilizing group comprising the N-terminus of the peptide drug and a second stabilizing group comprising the C-terminus of the peptide drug. Thus, in another preferred embodiment of the treatment method of the invention, the stabilized peptide drug comprises one or more prolines, more preferably a proline-proline dipeptide, attached to one or both termini of the peptide drug. For example, the peptide drug can be stabilized by covalent attachment of a Rop protein at one terminus, and by a proline or proline dipeptide at the other terminus; in another preferred embodiment, the peptide drug can be stabilized by proline dipeptides at each of the N-terminus and C-terminus. Alternatively, or in addition, the stabilized peptide drug used in the treatment method comprises an Xaa-Pro- or an Xaa-Pro-Pro- sequence at the N-terminus of the peptide drug, and/or a -Pro-Xaa or a -Pro-Pro-Xaa sequence at the C-terminus. Optionally, prior to administering the stabilized peptide drug, the treatment method can include a step comprising covalently linking a stabilizing group to one or both termini of the peptide drug to yield the stabilized peptide drug.

[0065] If desired, the unmodified peptide drug can conveniently be used as a control in a protease- or peptidase-resistance assay as described hereinabove to confirm that the stabilized peptide drug exhibits increased stability. Further, the therapeutic efficacy of the peptide drug is preferably preserved or enhanced in the stabilized peptide drug; modifications that reduce or eliminate the therapeutic efficacy of the peptide drug are easily detected and are to be avoided.

[0066] The present invention further includes a fusion protein comprising a four-helix bundle protein, preferably Rop protein, and a polypeptide. Preferably the polypeptide is bioactive; more preferably it is a bioactive peptide. The fusion protein of the invention can be used in any convenient expression vector known in the art for expression or overexpression of a peptide or protein of interest. Optionally, a cleavage site is present between four helix bundle protein and the polypeptide to allow cleavage, isolation and purification of the polypeptide. In one embodiment of the fusion protein, the four helix bundle protein is covalently linked at its C-terminus to the N-terminus of the polypeptide; in an alternative embodiment, the four helix bundle protein is covalently linked at its N-terminus to the C-terminus of the polypeptide. Fusion proteins of the invention, and expression vectors comprising nucleic acid sequences encoding fusion proteins wherein the nucleic acid sequences are operably linked to a regulatory control element such as a promoter, are useful for producing or overproducing any peptide or protein of interest.

EXAMPLES

[0067] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

Example I Construction and Characterization of a Highly Regulable Expression Vector, pLAC11, and its Multipurpose Derivatives, pLAC22 and pLAC33

[0068] A number of different expression vectors have been developed over the ears to facilitate the production of proteins in E. coli and related bacteria. Most of the routinely employed expression vectors rely on lac control in order to overproduce a gene of choice. While these vectors allow for overexpression of the gene product of interest, they are leaky due to changes that have been introduced into the lac control region and gene expression can never be shut off under repressed conditions, as described in more detail below. Numerous researchers have noticed this problem with the more popular expression vectors pKK223-3 (G. Posfai et al. Gene. 50: 63-67 (1986); N. Scrutton et al., Biochem J. 245: 875-880 (1987)), pKK233-2 (P. Beremand et al., Arch Biochem Biophys. 256: 90-100 (1987); K. Ooki et al., Biochemie. 76: 398-403 (1994)), pTrc99A (S. Ghosh, Protein Expr. Purif. 10: 100-106 (1997); J. Ranie et al., Mol. Biochem. Parasitol. 61: 159-169 (1993)), as well as the pET series (M. Eren et al., J. Biol. Chem. 264: 14874-14879 (1989); G. Godson, Gene 100: 59-64 (1991)).

[0069] The expression vector described in this example, pLAC11, was designed to be more regulable and thus more tightly repressible when grown under repressed conditions. This allows better regulation of cloned genes in order to conduct physiological experiments. pLAC11 can be used to conduct physiologically relevant studies in which the cloned gene is expressed at levels equal to that obtainable from the chromosomal copy of the gene in question. The expression vectors described here were designed utilizing the wild-type lac promoter/operator in order to accomplish this purpose and include all of the lac control region, without modification, that is contained between the start of the O3 auxiliary operator through the end of the O1 operator. As with all lac based vectors, the pLAC11 expression vector described herein can be turned on or off by the presence or absence of the gratuitous inducer IPTG. In experiments in which a bacterial cell contained both a null allele in the chromosome and a second copy of the wild-type allele on pLAC11, cells grown under repressed conditions exhibited the null phenotype while cells grown under induced conditions exhibited the wild-type phenotype. Thus the pLAC11 vector truly allows for the gene of interest to be grown under either completely repressed or fully induced conditions. Two multipurpose derivatives of pLAC11, pLAC22 and pLAC33, were also constructed to fulfill different experimental needs.

[0070] The vectors pLAC11, pLAC22 and pLAC33 were deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va., 20110-2209, USA, on Feb. 16, 1999, and assigned ATCC deposit numbers ATCC 207108, ATCC 207110 and ATCC 207109, respectively. It is nonetheless to be understood that the written description herein is considered sufficient to enable one skilled in the art to fully practice the present invention. Moreover, the deposited embodiment is intended as a single illustration of one aspect of the invention and is not to be construed as limiting the scope of the claims in any way.

[0071] Materials and Methods

[0072] Media. Minimal M9 media (6 g disodium phosphate, 3 g potassium phosphate, 1 g ammonium chloride, 0.5 g sodium chloride, distilled water to 1 L; autoclave; add 1 mL magnesium sulfate (1M) and 0.1 mL calcium chloride (1M); a sugar added to a final concentration of 0.2%; vitamins and amino acids as required for non-prototrophic strains) and rich LB media (10 g tryptone, 5 g yeast extract, 10 g sodium chloride, distilled water to 1 L; autoclave) were prepared as described by Miller (J. Miller, “Experiments in molecular genetics” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1972). The antibiotics ampicillin, kanamycin, streptomycin, and tetracycline (Sigma Chemical Company, St. Louis, Mo.) were used in rich media at a final concentration of 100, 40, 200, and 20 ug/ml, respectively. When used in minimal media, tetracycline was added at a final concentration of 10 ug/ml. 5-bromo-4-chloro-3-indoyl β-D-galactopyranoside (Xgal) was added to media at a final concentration of 40 ug/ml and unless otherwise noted. IPTG was added to media at a final concentration of 1 mM.

[0073] Chemicals and Reagents. When amplified DNA was used to construct the plasmids that were generated in this study, the PCR reaction was carried out using native Pfu polymerase from Stratagene (Cat. No. 600135). Xgal and IPTG were purchased from Diagnostic Chemicals Limited.

[0074] Bacterial Strains and Plasmids. Bacterial strains and plasmids are listed in Table 4. To construct ALS225, ALS224 was mated with ALS216 and streptomycin resistant, blue recombinants were selected on a Rich LB plate that contained streptomycin, Xgal, and IPTG. To construct ALS226, ALS224 was mated with ALS217 and streptomycin resistant, kanomycin resistant recombinants were selected on a Rich LB plate that contained streptomycin and kanamycin. To construct ALS515, ALS514 was mated with ALS216 and streptomycin resistant, blue recombinants were selected on a Rich LB plate that contained streptomycin, Xgal, and IPTG. To construct ALS527, ALS524 was mated with ALS224 and streptomycin resistant, tetracycline resistant recombinants were selected on a Rich LB plate that contained streptomycin and tetracycline. To construct ALS535, ALS533 was mated with ALS498 and tetracycline resistant recombinants were selected on a Minimal M9 Glucose plate that contained tetracycline, leucine and thiamine (B₁) (Sigma Chemical Company). To construct ALS533, a PI lysate prepared from E. coli strain K5076 (H. Miller et al., Cell 20: 711-719 (1980)) was used to transduce ALS224 and tetracycline resistant transductants were selected. TABLE 4 Bacterial strains and plasmids used in Example I Laboratory Original Name Name Genotype Source E. coli Strains ALS216 SE9100 araD139 Δ(lac)U169 thi f1bB5301 E. Altman et al., J. Biol. Chem. deoC7 ptsF25 rpsE/F′lacI^(q1) Z⁺ Y⁺ A⁺ 265: 18148-18153 (1990) ALS217 SE9100.1 araD139 Δ(lac)U169 thi flbB5301 S: Emr (Univ. of California, San Diego) deoC7 ptsF25 rpsE/F′ lacI^(q1) Z::Tn5 Y+ A+ ALS221 BL21(DE3) ompT hsdS(b) (R-M-) gal dcm F. Studier et al., J. Mol. Biol. 189: 113-130 (1986) ALS224 MC1061 araD139 Δ(araABOIC- M. Casadaban et al., J. Mol. Biol. leu)7679Δ(lac)X74 galU galK 138: 179-207 (1980). rpsL hsr− hsm+ ALS225 MC1061/F′lacI^(q1) Z⁺ Y⁺ A⁺ This example ALS226 MC1061/F′lacI^(q1) Z::Tn5 Y⁺ A⁺ This example ALS269 CSH27 F-trpA33 thi J. Miller, “Experiments in molecular genetics” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1972). ALS413 MG1655 E. coli wild-type F-λ- M. Guyer et al., Cold Spring Harbor Symp. Quant. Biol. 45:135-140 (1980). ALS498 JM101 supE thi Δ(lac-proAB)/F′ C. Yanisch-Perron et al., Gene. traD36 proA⁺B⁺ lacI^(q) Δ(lacZ)M15 33: 103-119 (1985) ALS514 NM554 MC1061 recA13 E. Raleigh et al., Nucl. Acids Res. 16: 1563-1575 (1988). ALS515 MC1061 recA13/F′lacI^(q1) Z⁺ Y⁺ A⁺ This example ALS524 XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 Stratagene (Cat. No. 200268) supE44 relA1 lac/F′ proAB lacI^(q)Δ(lacZ)M15 Tn10 ALS527 MC1061/F′ proAB This example lacI^(q) Δ(lacZ)M15 Tn10 ALS533 MC1061 proAB::Tn10 This example ALS535 MC1061 proAB::Tn10/F′lacI^(q) This example Δ(lacZ)M15 proA+B+ ALS598 CAG18615 zjb-3179::Tn10dKan lambda-rph-1 M. Singer et al., Microbiol. Rev. 53: 1-24 (1989). Plasmid Name Relevant Characteristics Source Plasmids pBH20 wild-type lac promoter/operator, K. Itakura et al., Science. 198: 1056-1063 (1977) Amp^(R), Tet^(R), colE1 replicon pBR322 Amp^(R), Tet^(R), colE1 replicon F. Bolivar et al., Gene. 2: 95-13 (1977) pET-21(+) T7 promoter/lac operator, lacI^(q), Novagen (Cat No. 69770-1) Amp^(R), colE1 replicon pGE226 wild-type recA gene, Amp^(R) J. Weisemann, et al., J. Bacteriol. 163: 748-755 (1985) pKK223-3 tac promoter/operator, Amp^(R), J. Brosius et al., Proc. Natl. Acad. Sci. USA. 81: 6929-6933 (1984) colE1 replicon pKK233-2 trc promoter/operator, Amp^(R), E. Amann et al., Gene. 40: 183-190 (1985) colE1 replicon pLysE T7 lysozyme, Cam^(R), P15A replicon F. Studier, J. Mol. Biol. 219: 37-44 (1991) pLysS T7 lysozyme, Cam^(R), P15A replicon F. Studier, J. Mol. Biol. 219: 37-44 (1991) pMS42l wild-type lac promoter/operator, D. Graña et al., Genetics. 120: 319-327 (1988) lacI^(q), Strep^(R), Spec^(R), SC101 replicon pTer7 wild-type lacZ coding region, R. Young (Texas A&M University) Amp^(R) pTrc99A trc promoter/operator, lacI^(q), E. Amann et al., Gene. 69: 301-315 (1988) Amp^(R), colE1 replicon pUC8 lac promoter/operator, Amp^(R), J. Vieira et al., Gene, 19: 259-268 (1982) colE1 replicon pXE60 wild-type TOL pWWO xylE gene, Amp^(R) J. Westpheling (Univ. of Georgia)

[0075] Construction of the pLAC11, pLAC22, and pLAC33 expression vectors. To construct pLAC11, primers #1 and #2 (see Table 5) were used to PCR amplify a 952 base pair (bp fragment from the plasmid pBH20 which contains the wild-type lac operon. Primer #2 introduced two different base pair mutations into the seven base spacer region between the Shine Dalgarno site and the ATG start site of the lacZ which converted it from AACAGCT to AAGATCT thus placing a Bgl II site in between the Shine Dalgarno and the start codon of the lacZ gene. The resulting fragment was gel isolated, digested with Pst I and EcoR I, and then ligated into the 3614 bp fragment from the plasmid pBR322ΔAvaI which had been digested with the same two restriction enzymes. To construct pBR322ΔAvaI, pBr322 was digested with AvaI, filled-in using Klenow, and then religated. To construct pLAC22, a 1291 bp Nco I, EcoR I fragment was gel isolated from pLAC21 and ligated to a 4361 bp Nco I, EcoR I fragment which was gel isolated from pBR322/NcoI. To construct pLAC21, primers #2 and #3 (see Table 5) were used to PCR amplify a 1310 bp fragment from the plasmid pMS421 which contains the wild-type lac operon as well as the lacIq repressor. The resulting fragment was gel isolated, digested with EcoR I, and then ligated into pBR322 which had also been digested with EcoR I. To construct pBR3221Nco I, primers #4 and #5 (see Table 5) were used to PCR amplify a 788 bp fragment from the plasmid pBR322. The resulting fragment was gel isolated, digested with Pst I and EcoR I, and then ligated into the 3606 bp fragment from the plasmid pBR322 which had been digested with the same two restriction enzymes. The pBR322/Nco I vector also contains added Kpn I and Sma I sites in addition to the new Nco I site. To construct pLAC33, a 2778 bp fragment was gel isolated from pLAC12 which had been digested with BsaB I and Bsa I and ligated to a 960 bp fragment from pUC8 which had been digested with Afl III, filled-in with Klenow, and then digested with Bsa I. To construct pLAC12, a 1310 bp Pst I, BamH I fragment was gel isolated from pLAC11 and ligated to a 3232 bp Pst I, BamH I fragment which was gel isolated from pBR322. TABLE 5 Primers employed to PCR amplify DNA fragments that were used in the construction of the various plasmids described in Example 1 pLAC11 and pLAC22 1 (for) GTT GCC ATT GCT GCA GGC AT (SEQ ID NO:6) 2 (rev) ATT GAA TTC ATA AGA TCT TTC CTG TGT GAA ATT GTT ATC CGC (SEQ ID NO:7) 3 (for) ATT GAA TTC ACC ATG GAC ACC ATC GAA TGG TGC AAA A (SEQ ID NO:8)   pBR322/Nco I 4 (for) GTT GTT GCC ATT GCT GCA G (SEQ ID NO:9) 5 (rev) TGT ATG AAT TCC CGG GTA CCA TTG TTG AAG ACG AAA GGG CCT C (SEQ ID NO:10)   Bgl II-lacZ-Hind III 6 (for) TAC TAT AGA TCT ATG ACC ATG ATT ACG GAT TCA CTG (SEQ ID NO:11) 7 (rev) TAC ATA AAG CTT GGC CTG CCC GGT TAT TAT TAT TTT (SEQ ID NO:12)   Pst I-lacZ-Hind III 8 (for) TAT CAT CTG CAG AGG AAA CAG CTA TGA CCA TGA TTA CGG ATT CAC TG (SEQ ID NO:13) 9 (rev) TAC ATA CTC GAG CAG GAA AGC TTG GCC TGC CCG GTT ATT ATT ATT TT (SEQ ID NO:14)   BamH 1-lacZ-Hind III (also uses primer #9) 10 (for) TAT CAT GGA TCC AGG AAA CAG CTA TGA CCA TGA TTA CGG ATT CAC TG (SEQ ID NO:15)   Bgl II-recA-Hind III 11 (for) TAC TAT AGA TCT ATG GCT ATC GAC GAA AAC AAA CAG (SEQ ID NO:16) 12 (rev) ATA TAT AAG CTT TTA AAA ATC TTC GTT AGT TTC TGC TAC G (SEQ ID NO:17)   BamH 1-xylE-EcoR I 13 (for) TAC TAT AGA TCT ATG AAC AAA GGT GTA ATG CGA CC (SEQ ID NO:18) 14 (rev) ATT AGT GAA TTC GCA CAA TCT CGT CAA TAA GTC GT (SEQ ID NO:19)

[0076] In Table 5 the regions of the primers that are homologous to the DNA target template are indicated with a dotted underline, while the relevant restriction sites are indicated with a solid underline. All primers are listed in the 5′→3′ orientation.

[0077] Compilation of the DNA sequences for the pLAC11, pLAC22, and pLAC33 expression vectors. All of the DNA that is contained in the pLAC11, pLAC22, and, pLAC33 vectors has been sequenced.

[0078] The sequence for the pLAC11 vector which is 4547 bp can be compiled as follows: bp 1-15 are AGATCTTATGAATTC (SEQ ID NO:20) from primer #2 (Table 5); bp 16-1434 are bp 4-1422 from pBR322 (GenBank Accession #J01749); bp 1435-1442 are TCGGTCGG, caused by filling in the Ava I site in pBR322ΔAvaI; bp 1443-4375 is bp 1427-4359 from pBR322 (GenBank Accession #J01749); and bp 4376-4547 are bp 1106-1277 from the wild-type E. coli lac operon (GenBank Accession #J01636).

[0079] The sequence for the pLAC22 vector which is 5652 bp can be compiled as follows: bp 1-15 are AGATCTTATGAATTC (SEQ ID NO:21) from primer #2 (Table 5); bp 16-4370 are bp 4-4358 from pBR322 (GenBank Accession #J01749); bp 4371-4376 is CCATGG which is the Nco I site from pBR322/Nco I; and bp 4377-5652 are bp 2-1277 from the wild-type E. coli lac operon (GenBank Accession #J01636), except that bp #4391 of the pLAC22 sequence or bp#16 from the wild-type E. coli lac operon sequence has been changed from a “C” to a “T” to reflect the presence of the lacIq mutation (J. Brosius et al., Proc. Natl. Acad. Sci. USA. 81:6929-6933 (1984)).

[0080] The sequence for the pLAC33 vector which is 3742 bp can be compiled as follows: bp 1-15 is AGATCTTATGAATTC (SEQ ID NO:22) from primer #2 (Table 5); bp 16-1684 are bp 4-1672 from pBR322 (GenBank Accession #J01749); bp 1685-2638 are bp 786-1739 from pUC8 (GenBank Accession #L09132); bp 2639-3570 are bp 3428-4359 from pBR322 (GenBank Accession #J01749); and bp 3571-3742 are bp 1106-1277 from the wild-type E. coli lac operon (GenBank Accession #J01636). In the maps for these vectors the ori is identified as per Balbás (P. Balbás et al., Gene. 50:340 (1986)) while the lacPO is indicated starting with the O3 auxiliary operation and ending with the O1 operator as per Müller-Hill (B. Müller-Hill, The lac Operon: A Short History of a Genetic Paradigm, Walter de Gruyter, Berlin, Germany (1996)).

[0081] Construction of the pLAC11-, pLAC22-, pLAC33-, pKK223-3-, pKK233-2-, pTrc99A-, and pET-21(+)-lacZ constructs. To construct pLAC11-lacZ, pLAC22-lacZ, and pLAC33-lacZ, primers #6 and #7 (see Table 5) were used to PCR amplify a 3115 bp fragment from the plasmid pTer7 which contains the wild-type lacZ gene. The resulting fragment was gel isolated, digested with Bgl II and Hind III, and then ligated into the pLAC11, pLAC22, or pLAC33 vectors which had been digested with the same two restriction enzymes. To construct pKK223-3-lacZ and pKK233-2-lacZ, primers #8 and #9 (see Table 5) were used to PCR amplify a 3137 bp fragment from the plasmid pTer7. The resulting fragment was gel isolated, digested with Pst I and Hind III, and then ligated into the pKK223-3 or pKK233-2 vectors which had been digested with the same two restriction enzymes. To construct pTrc99A-lacZ and pET-21(+)-lacZ, primers #9 and #10 (see Table 5) were used to PCR amplify a 3137 bp fragment from the plasmid pTer7. The resulting fragment was gel isolated, digested with BamH I and Hind III, and then ligated into the pTrc99A or pET-21(+) vectors which had been digested with the same two restriction enzymes.

[0082] Construction of the pLAC11-recA and xylE constructs. To construct pLAC11-recA, primers #11 and #12 (see Table 5) were used to PCR amplify a 1085 bp fragment from the plasmid pGE226 which contains the wild-type recA gene. The resulting fragment was gel isolated, digested with Bgl II and Hind III, and then ligated into the pLAC11 vector which had been digested with the same two restriction enzymes. To construct pLAC11-xylE, primers #13 and #14 (see Table 5) were used to PCR amplify a 979 bp fragment from the plasmid pXE60 which contains the wild-type Pseudomonas putida xylE gene isolated from the TOL pWWO plasmid. The resulting fragment was gel isolated, digested with Bgl II and EcoR I, and then ligated into the pLAC11 vector which had been digested with the same two restriction enzymes.

[0083] Assays. β-galactosidase assays were performed as described by Miller (J. Miller, “Experiments in molecular genetics” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1972)), while catechol 2,3-dioxygenase (catO₂ase) assays were performed as described by Zukowski, et. al. (M. Zukowski et al., Proc. Natl. Acad. Sci. U.S.A. 80:1101-1105 (1983)).

[0084] Results

[0085] Construction and features of pLAC11, pLAC22, and pLAC33. Plasmid maps that indicate the unique restriction sites, drug resistances, origin of replication, and other relevant regions that are contained in pLAC11, pLAC22, and pLAC33 are shown in FIGS. 2, 3 and 4, respectively. pLAC11 was designed to be the most tightly regulable of these vectors. It utilizes the ColE1 origin of replication from pBR322 and LacI repressor is provided in trans from either an episome or another compatible plasmid. pLAC22 is very similar to pLAC11, however, it also contains lacIq, thus a source of LacI does not have to be provided in trans. pLAC33 is a derivative of pLAC11 which utilizes the mutated ColE1 origin of replication from pUC8 (S. Lin-Chao et al., Mol. Micro. 6:3385-3393 (1992)) and thus pLAC33's copy number is significantly higher than pLAC 1I and is comparable to that of other pUC vectors. Because the cloning regions of these three vectors are identical, cloned genes can be trivially shuffled between and among them depending on the expression demands of the experiment in question.

[0086] To clone into pLAC11, pLAC22, or pLAC33, PCR amplification is performed with primers that are designed to introduce unique restriction sites just upstream and downstream of the gene of interest. Usually a Bgl II site is introduced immediately in front of the ATG start codon and an EcoR I site is introduced immediately following the stop codon. An additional 6 bases is added to both ends of the oligonucleotide in order to ensure that complete digestion of the amplified PCR product will occur. After amplification the double-stranded (ds) DNA is restricted with Bgl II and EcoR I, and cloned into the vector which has also been restricted with the same two enzymes. If the gene of interest contains a Bgl II site, then BamH I or Bcl I can be used instead since they generate overhangs which are compatible with Bgl II. If the gene of interest contains an EcoR I site, then a site downstream of EcoR I in the vector (such as Hind III) can be substituted.

[0087] Comparison of pLAC11, pLAC22, and pLAC33, to other expression vectors. In order to demonstrate how regulable the pLAC11, pLAC22, and pLAC33 expression vectors were, the wild-type lacZ gene was cloned into pLAC11, pLAC22, pLAC33, pKK223-3, pKK233-2, pTrc99A, and pET-21(+). Constructs which required an extraneous source of LacI for their repression were transformed into ALS225, while constructs which contained a source of LacI on the vector were transformed into ALS224. pET-21(+) constructs were transformed into BL21 because they require T7 RNA polymerase for their expression. Four clones were chosen for each of these seven constructs and β-galactosidase assays were performed under repressed and induced conditions. Rich Amp overnights were diluted 1 to 200 in either Rich Amp Glucose or Rich Amp IPTG media and grown until they reached mid-log (OD₅₅₀=0.5). In the case of pET-21(+) the pLysE and pLysS plasmids, which make T7 lysozyme and thus lower the amount of available T7 polymerase, were also transformed into each of the constructs. Table 6 shows the results of these studies and also lists the induction ratio that was determined for each of the expression vectors. As the data clearly indicates, pLAC11 is the most regulable of these expression vectors and its induction ratio is close to that which can be achieved with the wild-type lac operon. The vector which yielded the lowest level of expression under repressed conditions was pLAC11, while the vector which yielded the highest level of expression under induced conditions was pLAC33. TABLE 6 β-galactosidase levels obtained in different expression vectors grown under either repressed or induced conditions # of Miller Units Observed Repressed Induced Fold Vector Source Conditions Conditions Induction pLAC11 F′ 19 11209 590X  pLAC22 Plasmid 152 13315 88X pLAC33 F′ 322 23443 73X pKK223-3 F′ 92 11037 120X  pKK233-2 F′ 85 10371 122X  pTrc99A Plasmid 261 21381 82X pET-21(+) Plasmid 2929 16803  6X pET-21(+)/pLysE Plasmid 4085 19558  5X pET-21(+)/pLysS Plasmid 1598 20268 13X

[0088] The average values obtained for the four clones that were tested from each vector are listed in the table. Standard deviation is not shown but was less than 5% in each case. Induction ratios are expressed as the ratio of enzymatic activity observed at fully induced conditions versus fully repressed conditions. The plasmid pLysE yielded unexpected results; it was expected to cause lower amounts of lacZ to be expressed from pET-21(+) under repressed conditions and, instead, higher amounts were observed. As a result, both pLysE and pLysS were restriction mapped to make sure that they were correct.

[0089] Demonstrating that pLAC11 constructs can be tightly regulated. pLAC11 was designed to provide researchers with an expression vector that could be utilized to conduct physiological experiments in which a cloned gene is studied under completely repressed conditions where it is off or partially induced conditions where it is expressed at physiologically relevant levels. FIG. 5 demonstrates how a pLAC11-lacZ construct can be utilized to mimic chromosomally expressed lacZ that occurs under various physiological conditions by varying the amount of IPTG inducer that is added. ALS226 cells containing pLAC11-lacZ were grown to mid-log in rich media that contained varying amounts of IPTG and then β-galactosidase activity was assayed. Also indicated in the graph are the average β-galactosidase activities obtained for strains with a single chromosomal copy of the wild-type lacZ gene that were grown under different conditions.

[0090] To demonstrate just how regulable pLAC11 is, the recA gene was cloned into the pLAC11 vector and transformed into cells which contained a null recA allele in the chromosome. As the results in Table 7 clearly shows, recombination can not occur in a host strain which contains a nonfunctional RecA protein and thus P1 lysates which provide a Tn10dKan transposon can not be used to transduce the strain to Kan^(R) at a high frequency. recA⁻ cells which also contain the pLAC11-recA construct can be transduced to Kan^(R) at a high frequency when grown under induced conditions but can not be transduced to Kan^(R) when grown under repressed conditions. TABLE 7 The recombination (−) phenotype of a recA null mutant strain can be preserved with a pLAC11-rec/4 (wild-type) construct under repressed conditions Repressed Conditions Induced Conditions Number of Kan^(R) Number of Kan^(R) Strain transductants transductants ALS225 (recA⁺) 178,000 182,000 ALS514 (recA⁻) 5 4 ALS515 4 174,000 (recA⁻ pCyt-3-recA)

[0091] The data presented in Table 7 are the number of Kan^(R) transductants that were obtained from the different MC1061 derivative strains when they were transduced with a P1 lysate prepared from strain ALS598 which harbored a Tn10dKan transposon insertion. Overnights were prepared from each of these strains using either rich medium to which glucose was added at a final concentration of 0.2% (repressed conditions) or rich medium to which IPTG was added at a final concentration of 1 mM (induced conditions). The overnights were then diluted 1 to 10 into the same medium which contained CaCl₂ added to a final concentration of 10 mM and aerated for two hours to make them competent for transduction with P1 phage. Cells were then spectrophotometrically normalized and aliquots of 5 OD₅₅₀ cell equivalents in a volume of approximately 0.1 ml were transduced with 0.1 ml of concentrated P1 lysate as well as 0.1 ml of P1 lysates that had been diluted to 10⁻¹, 10⁻², or 10⁻³. 0.2 ml of 0.1M Sodium Citrate was added to the cell/phage mixtures and 0.2 ml of the final mixtures were plated onto Rich Kanamycin plates and incubated overnight at 37° C. The total number of Kan^(R) colonies were then counted. ALS225 recA⁺ data points were taken from the transductions which used the 10⁻³ diluted phage, while ALS5 14 recA⁻ data points were taken from the transductions which used the concentrated phage. The data points for ALS5 15 recA⁻ pCyt-3-recA grown under repressed conditions were taken from the transductions which used the concentrated phage, while the data points for ALS515 recA⁻ pCyt-3-recA grown under induced conditions were taken from the transductions which used the 10⁻³ diluted phage.

[0092] Testing various sources of LacI for trans repression of pLAC11. Because pLAC11 was designed to be used with an extraneous source of LacI repressor, different episomal or plasmid sources of LacI which are routinely employed by researchers were tested. Since one of the LacI sources also contained the lacZ gene, a reporter construct other than pLAC11-lacZ was required and thus a pLAC11-xylE construct was engineered. Table 8 shows the results of this study.

[0093] All of the LacI sources that were tested proved to be adequate to repress expression from pLAC11, however, some were better than others. The basal level of expression that was observed with F's which provided lacIq¹ or with the plasmid pMS421 which provided lacIq at approximately six copies per cell was lower than the basal level of expression that was observed with F's which provided lacIq all three times that the assay was run. Unfortunately, however, the xylE gene could not be induced as high when lacIq¹ on a F′ or lacIq on a plasmid was used as the source of Lac repressor. TABLE 8 Catechol 2,3-dioxygenase levels obtained for a pLAC11-xylE construct when Lac represser is provided by various sources Catechol 2,3-dioxygenase activity in milliunits/mg Repressed Induced Strain Source of LacI Conditions Conditions ALS224 None 32.7 432.8 ALS535 F′lacI^(q) Δ(lacZ)M15 .3 204.4 proA+B+ Tn10 ALS527 F′lacI^(q)Δ(lacZ)M15 .3 243.3 proA+B+ ALS227 pMS421 lacI^(q) .2 90.9 ALS225 F′lacI^(q1) Z⁺ Y⁺ A⁺ .2 107.4 ALS226 F′lacI^(q1) Z::Tn5 Y⁺ A⁺ .2 85.1

[0094] The wild-type xylE gene was cloned into the pLAC11 vector and the resulting pLAC11-xylE construct was then transformed into each of the MC1061 derivative strains listed in the table. Rich overnights were diluted 1 to 200 in either Rich Glucose or Rich IPTG media and grown until they reached mid-log (OD₅₅₀=0.5). Cell extracts were then prepared and catechol 2,3-dioxygenase assays were performed as described by Zukowski, et. al (Proc. Natl. Acad. Sci. U.S.A. 80:1101-1105 (1983)). The average values obtained in three different experiments are listed in the table. Standard deviation is not shown but was less than 10% in each case.

[0095] Discussion

[0096] Most of the routinely employed expression vectors rely on lac control in order to overproduce a gene of choice. The lac promoter/operator functions as it does due to the interplay of three main components. First, the wild-type lac −10 region (TATGTT) is very weak. c-AMP activated CAP protein is able to bind to the CAP site just upstream of the −35 region which stimulates binding of RNA polymerase to the weak −10 site. Repression of the lac promoter is observed when glucose is the main carbon source because very little c-AMP is present which results in low amounts of available c-AMP activated CAP protein. When poor carbon sources such as lactose or glycerol are used, c-AMP levels rise and large amounts of c-AMP activated CAP protein become available. Thus induction of the lac promoter can occur. Second, Lac repressor binds to the lac operator. Lac repressor can be overcome by allolactose which is a natural byproduct of lactose utilization in the cell, or by the gratuitous inducer, IPTG. Third, the lac operator can form stable loop structures which prevents the initiation of transcription due to the interaction of the Lac repressor with the lac operator (O1) and one of two auxiliary operators, O2 which is located downstream in the coding region of the lacZ gene, or O3 which is located just upstream of the CAP binding site.

[0097] While binding of Lac repressor to the lac operator is the major effector of lac regulation, the other two components are not dispensable. However, most of the routinely used lac regulable vectors either contain mutations or deletions which alter the affect of the other two components. The pKK223-3 (J. Brosius et al., Proc. Natl. Acad. Sci. USA. 81:6929-6933 (1984)), pKK233-2 (E. Amann et al., Gene. 40:183-190 (1985)), pTrc99A (E. Amann et al., Gene. 69:301-315 (1988)), and pET family of vectors (F. Studier, Method Enzymol. 185:60-89 (1990)) contain only the lac operator (O1) and lack both the CAP binding site as well as the O3 auxiliary operator. pKK223-3, pKK233-2, and pTrc99 use a trp-lac hybrid promoter that contains the trp −35 region and the lacUV5-10 region which contains a strong TATAAT site instead of the weak TATGTT site. The pET family of vectors use the strong T7 promoter. Given this information, perhaps it is not so surprising researchers have found it is not possible to tightly shut off genes that are cloned into these vectors.

[0098] The purpose of the studies described in Example I was to design a vector which would allow researchers to better regulate their cloned genes in order to conduct physiological experiments. The expression vectors described herein were designed utilizing the wild-type lac promoter/operator in order to accomplish this purpose and include all of the lac control region, without modification, that is contained between the start of the O3 auxiliary operator through the end of the O1 operator. As with all lac based vectors, the pLAC11, pLAC22, and pLAC33 expression vectors can be turned on or off by the presence or absence of the gratuitous inducer IPTG.

[0099] Because the new vector, pLAC11, relies on the wild-type lac control region from the auxiliary lac O3 operator through the lac O1 operator, it can be more tightly regulated than the other available expression vectors. In direct comparison studies with pKK223-3, pKK233-2, pTrc99A, and pET-21(+), the lowest level of expression under repressed conditions was achievable with the pLAC11 expression vector. Under fully induced conditions, pLAC11 expressed, lacZ protein that was comparable to the levels achievable with the other expression vectors. Induction ratios of 1000× have been observed with the wild-type lac operon. Of all the expression vectors that were tested, only pLAC11 yielded induction ratios which were comparable to what has been observed with the wild-type lac operon. It should be noted that the regulation achievable by pLAC11 may be even better than the data in Table 6 indicates. Because lacZ was used in this test, the auxiliary lac O₂ operator which resides in the coding region of the lacZ gene was provided to the pKK223-3, pKK233-2, pTrc99A, and pET-21(+) vectors which do not normally contain either the O2 or O3 auxiliary operators. Thus the repressed states that were observed in the study in Table 6 are probably lower than one would normally observe with the pKK223-3, pKK233-2, pTrc99A, and pET-21(+) vectors.

[0100] To meet the expression needs required under different experimental circumstances, two additional expression vectors which are derivatives of pLAC11 were designed. pLAC22 provides lacIq on the vector and thus unlike pLAC11 does not require an extraneous source of LacI for its repression. pLAC33 contains the mutated ColE1 replicon from pUC8 and thus allows proteins to be expressed at much higher levels due to the increase in the copy number of the vector. Of all the expressions that were evaluated in direct comparison studies, the highest level of protein expression under fully induced conditions was achieved using the pLAC33 vector. Because the cloning regions are identical in pLAC11, pLAC22, find pLAC33, genes that are cloned into one of these vectors can be trivially subcloned into either of the other two vectors depending on experimental circumstances. For physiological studies, pLAC11 is the best suited of the three vectors. If, however, the bacterial strain of choice can not be modified to introduce elevated levels of Lac repressor protein which can be achieved by F's or compatible plasmids that provide lacIq or lacIq¹, the pLAC22 vector can be utilized. If maximal overexpression of a gene product is the goal, then the pLAC33 vector can be utilized.

[0101] Numerous experiments call for expression of a cloned gene product at physiological levels; i.e., at expression levels that are equivalent to the expression levels observed for the chromosomal copy of the gene. While this is not easily achievable with any of the commonly utilized expression vectors, these kinds of experiments can be done with the pLAC11 expression vector. By varying the IPTG concentrations, expression from the pLAC11 vector can be adjusted to match the expression levels that occur under different physiological conditions for the chromosomal copy of the gene. In fact, strains which contain both a chromosomal null mutation of the gene in question and a pLAC11 construct of the gene preserve the physiological phenotype of the null mutation under repressed conditions.

[0102] Because the use of Lac repressor is an essential component of any expression vector that utilizes the lac operon for its regulation, the ability of different source of LacI to repress the pLAC11 vector was also investigated. Researchers have historically utilized either lacIq constructs which make 10 fold more Lac repressor than wild-type lacI or lacIq¹ constructs which make 100 fold more Lac repressor than wild-type lacI (B. Müller-Hill, Prog. Biophys. Mol. Biol. 30:227-252 (1975)). The greatest level of repression of pLAC11 constructs could be achieved using F's which provided approximately one copy of the lacIq¹ gene or a multicopy compatible plasmid which provided approximately six copies of the lacIq gene. However, the induction that was achievable using these lacI sources was significantly lower than what could be achieved when F's which provided approximately one copy of the lacIq gene were used to repress the pLAC11 construct. Thus if physiological studies are the goal of an investigation, then F's which provide approximately one copy of the lacIq¹ gene or a multicopy compatible plasmid which provides approximately six copies of the lacIq gene can be used to regulate the pLAC11 vector. However, if maximal expression is desired, then F's which provide approximately one copy of the lacIq gene can be utilized. Alternatively, if a bacterial strain can tolerate prolonged overexpression of an expressed gene and overexpression of a gene product is the desired goal, then maximal expression under induced conditions is obtained when a bacteria strain lacks any source of Lac repressor.

Example II An in vivo Approach for Generating Novel Bioactive Peptides that Inhibit the Growth of E. coli

[0103] A randomized oligonucleotide library containing sequences capable of encoding peptides containing up to 20 amino acids was cloned into pLAC11 (Example I) which allowed the peptides to either be tightly turned off or overproduced in the cytoplasm of E. coli. The randomized library was prepared using a [NNN] codon design instead of either the [NN(G,T)] or [NN(G,C)] codon design used by most fusion-phage technology researchers. [N(G,T)] or [NN(G,C)] codons have been widely used instead of [NNN] codons to eliminate two out of the three stop codons, thus increasing the amount of full-length peptides that can be synthesized without a stop codon (J. Scott et al., Science 249:386-390 (1990); J. Delvin et al., Science 249:404-406 (1990); S. Cwirla et al., Proc. Nat'l. Acad. Sci. U.S.A. 87:6378-6382 (1990)). However, the [NN(G,T)] and [NN(G,C)] oligonucleotide codon schemes eliminate half of the otherwise available codons and, as a direct result, biases the distribution of amino acids that are generated. Moreover, the [M(G,T)] and [NN(G,C)] codon schemes drastically affect the preferential codon usage of highly expressed genes and removes a number of the codons which are utilized by the abundant tRNAs that are present in E. coli (H. Grosjean et al., Gene. 18: 199-209 (1982); T. Ikemura, J. Mol. Biol. 151: 389-409 (1981)).

[0104] Of the 20,000 peptides screened in this Example, 21 inhibitors of cell growth were found which could prevent the growth of E. coli on minimal media. The top twenty inhibitor peptides were evaluated for strength of inhibition, and the putative amino acid sequences of the top 10 “anchorless” inhibitor peptides were examined for commonly shared features or motifs.

[0105] Materials and Methods

[0106] Media. Rich LB and minimal M9 media used in this study was prepared as in Example I. Ampicillin was used in rich media at a final concentration of 100 ug/ml and in minimal media at a final concentration of 50 ug/ml. IPTG was added to media at a final concentration of 1 mM.

[0107] Chemicals and Reagents. Extension reactions were carried out using Klenow from New England Biolabs while ligation reactions were performed using T4 DNA Ligase from Life Sciences. IPTG was obtained from Diagnostic Chemicals Limited.

[0108] Bacterial Strains and Plasmids. ALS225, which is MC1061/F′lacIq¹Z+Y+A+ (see Example I), was the E. coli bacterial strain used in this Example. The genotype for MC1061 is araD139 Δ(araABOIC-leu)7679 Δ(lac)X74 galU galK rpsL hsr− hsm+ (M. Casadaban et al., J. Mol. Biol. 138:179-207 (1980)). pLAC11, a highly regulable expression vector, is described in Example I.

[0109] Generation of the Randomized Peptide Library. The 93 base oligonucleotide 5′ TAC TAT AGA TCT ATG (NNN)₂₀ TAA TAA GAA TTC TCG ACA 3′ (SEQ ID NO: 23), where N denotes an equimolar mixture of the nucleotides A, C, G, or T, was synthesized with the trityl group and subsequently purified with an OPC cartridge using standard procedures. The complementary strand of the 93 base oligonucleotide was generated by an extension/fill-in reaction with Klenow using an equimolar amount of the 18 base oligonucleotide primer 5′ TGT CGA GAA TTC TTA TTA 3′ (SEQ ID NO: 24). After extension, the resulting ds-DNA was purified using a Promega DNA clean-up kit and restricted with EcoR 1 and Bgl II (Promega, Madison, Wis.). The digested DNA was again purified using a Promega DNA clean-up kit and ligated to pLAC11 vector which had been digested with the same two restriction enzymes. The resulting library was transformed into electrocompetent ALS225 E. coli cells under repressed conditions (LB, ampicillin, plus glucose added to 0.2%).

[0110] Screening of Transformants to Identify Inhibitor Clones. Transformants were screened to identify any that could not grow on minimal media when the peptides were overproduced. Using this scheme, any transformant bacterial colony that overproduces a peptide that inhibits the production or function of a protein necessary for growth of that transformant on minimal media will be identified. Screening on minimal media, which imposes more stringent growth demands on the cell, will facilitate the isolation of potential inhibitors from the library. It is well known that growth in minimal media puts more demands on a bacterial cell than growth in rich media as evidenced by the drastically reduced growth rate; thus a peptide that adversely affects cell growth is more likely to be detected on minimal media. Screening was carried out using a grid-patching technique. Fifty clones at a time were patched onto both a rich repressing plate (LB Amp glucose) and a minimal inducing plate (M9 glycerol Amp IPTG) using an ordered grid. Patches that do not grow are sought because presumably these represent bacteria that are being inhibited by the expressed bioactive peptide. To verify that all of the inhibitors were legitimate, plasmid DNA was made from each inhibitory clone (QIA Prep Spin Miniprep kit; Qiagen Cat. No. 27104) and transformed into a fresh background (ALS225 cells), then checked to confirm that they were still inhibitory on plates and that their inhibition was dependent on the presence of the inducer, IPTG.

[0111] Growth Rate Analysis in Liquid Media. Inhibition strength of the peptides was assessed by subjecting the inhibitory clones to a growth rate analysis in liquid media. To determine the growth rate inhibition, starting cultures of both the peptides to be tested and a control strain which contains pLAC11 were diluted from a saturated overnight culture to an initial OD₅₅₀ of ˜0.01. All cultures were then induced with 1 mM IPTG and OD₅₅₀ readings were taken until the control culture reached an OD₅₅₀ of ˜0.5. The hypothetical data in Table 9 shows that when the control strain reaches an OD₅₅₀ of about 0.64 (at about 15 hours), a strain which contains a peptide that inhibits the growth rate at 50% will only have reached an OD₅₅₀ of only about 0.08. Thus, the growth of a 50% inhibited culture at 15 hours (i.e., the OD₅₅₀ at 15 hours, which is proportional to the number of cells in a given volume of culture) is only about 12.5% (that is, 0.08/0.64×100) of that of a control strain after the same amount of time, and the inhibitor peptide would thus have effectively inhibited the growth of the culture (as measured by the OD₅₅₀ at the endpoint) by 87.5% (=100%-12.5%). TABLE 9 Hypothetical data from a peptide that inhibits growth rate at 30%, 50% and 70% OD550 readings OD550 readings on a culture which on a control contains a peptide that inhibits Time in culture which the growth rate at . . . hours contains pLAC11 25% 50% 75% 0 .010 .010 .010 .010 2.5 .020 .017 .015 .012 5 .040 .028 .020 .014 7.5 .080 .047 .030 .017 10 .160 .079 .040 .020 12.5 .320 .133 .060 .024 15 .640 .226 .080 .028

[0112] An example is shown in FIG. 6, wherein ALS225 cells containing the pLAC11 vector (control), and either the one day inhibitor pPep1 or the two day inhibitor pPep12 (see below), were grown in minimal M9 glycerol media with IPTG added to 1 mM. OD₅₅₀ readings were then taken hourly until the cultures had passed log phase. Growth rates were determined by measuring the spectrophotometric change in OD₅₅₀ per unit time within the log phase of growth. The inhibition of the growth rate was then calculated for the inhibitors using pLAC11 as a control.

[0113] Sequencing the Coding Regions of the Inhibitor Peptide Clones. The forward primer 5′ TCA TTA ATG CAG CTG GCA CG 3′ (SEQ ID NO: 25) and the reverse primer 5′ TTC ATA CAC GGT GCC TGA CT 3′ (SEQ ID NO: 26)

[0114] were used to sequence both strands of the top ten “anchorless” inhibitor peptide clones identified by the grid-patching technique. If an error-free consensus sequence could not be deduced from these two sequencing runs, both strands of the inhibitor peptide clones in question were resequenced using the forward primer 5′ TAG CTC ACT CAT TAG GCA CC 3′ (SEQ ID NO: 27) and the reverse primer 5′ GAT GAC GAT GAG CGC ATT GT 3′ (SEQ ID NO: 28). The second set of primers were designed to anneal downstream of the first set of primers in the pLAC11 vector.

[0115] Generating Antisense Derivatives of the Top Five “Anchorless” Inhibitor Clones. Oligonucleotides were synthesized which duplicated the DNA insert contained between the Bgl II and EcoR I restriction sites for the top five “anchorless” inhibitor peptides as shown in Table 12 with one major nucleotide change. The “T” of the ATG start codon was changed to a “C” which resulted in a ACG which can not be used as a start codon. The oligonucleotides were extended using the same 18 base oligonucleotide primer that was used to build the original peptide library. The resulting ds-DNA was then restricted, and cloned into pLAC11 exactly as described in the preceding section “Generating the randomized peptide library”. The antisense oligonucleotides that were used are as follows: pPep1(antisense): 5′ TAC TAT AGA TCT ACG GTC ACT GAA TTT TGT (SEQ ID NO: 29) GGC TTG TTG GAC CAA CTG CCT TAG TAA TAG TGG AAG GCT GAA ATT AAT AAG AAT TCT CGA CA 3′; pPep5(antisense): 5′ TAC TAT AGA TCT ACG TGG CGG GAC TCA TGG (SEQ ID NO: 30) ATT AAG GGT AGG GAC GTG GGG TTT ATG GGT TAA AAT AGT TTG ATA ATA AGA ATT CTC GAC A 3′ pPep12(antisense): 5′ TAC TAT AGA TCT ACG AAC GGC CGA ACC AAA (SEQ ID NO: 31) CGA ATC CGG GAC CCA CCA GCC GCC TAA ACA GCT ACC AGC TGT GGT AAT AAG AAT TCT CGA CA 3′ pPep13(antisense): 5′ TAC TAT AGA TCT ACG GAC CGT GAA GTG ATG (SEQ ID NO: 32) TGT GCG GCA AAA CAG GAA TGG AAG GAA CGA ACG CCA TAG GCC GCG TAA TAA GAA TTC TCG ACA 3′ pPep19(antisense): 5′ TAC TAT AGA TCT ACG AGG GGC GCC AAC TAA (SEQ ID NO: 33) GGG GGG GGG AAG GTA TTT GTC CCG TGC ATA ATC TCG GGT GTT GTC TAA TAA GAA TTC TCG ACA 3′

[0116] Results

[0117] Identifying and Characterizing Inhibitor Peptides from the Library. Approximately 20,000 potential candidates were screened as described hereinabove, and 21 IPTG-dependent growth inhibitors were isolated. All the inhibitors so identified were able to prevent the growth of the E. coli bacteria at 24 hours, and three of the 21 inhibitors were able to prevent the growth of the E. coli bacteria at 48 hours, using the grid patching technique. These three inhibitors were classified as “two day” inhibitors; the other 18 were classified as “one-day” inhibitors.

[0118] Results from the growth rate analysis for candidate peptide inhibitors are shown in Table 10. The % inhibition of the growth rate was calculated by comparing the growth rates of cells that contained induced peptides with the growth rate of cells that contained the induced pLAC11 vector. Averaged values of three independent determinations are shown. TABLE 10 Ability of the Inhibitor Peptides to Inhibit Cell Growth Inhibitor Type % Inhibition Inhibitor Type % Inhibition pLAC11 — 0 Ppep11 1 Day 22 (control) pPep1 1 Day 25 Ppep12 2 Day 82 pPep2 1 Day 23 Ppep13 1 Day 28 pPep3 2 Day 80 Ppep14 2 Day 71 pPep4 1 Day 21 Ppep15 1 Day 23 pPep5 1 Day 24 Ppep16 1 Day 24 pPep6 1 Day 27 Ppep17 1 Day 28 pPep7 1 Day 26 PPep18 1 Day 24 pPep8 1 Day 29 pPep19 1 Day 29 pPep9 1 Day 22 pPep20 1 Day 19 pPep10 1 Day 24 pPep21 1 Day 23

[0119] Of the 21 peptides that were tested, the one-day inhibitor peptides inhibited the bacterial growth rate at a level of approximately 25%, while the two-day inhibitor peptides inhibited the bacterial growth rate at levels greater than 75%. As can be seen from the hypothetical data in Table 9, a one-day inhibitor which inhibited the growth rate at 25% would have only reached an OD₅₅₀ of 0.226 when the control strain reached an OD₅₅₀ of 0.64. At that point in time, the growth of the culture that is inhibited by a one-day inhibitor (as measured by the end-point OD₅₅₀) will only be only 35.3% of that of a control strain at that point; thus the inhibitor peptide would have effectively inhibited the growth of the culture by 64.7%. A two-day inhibitor which inhibited the growth rate at 75% would have only reached an OD₅₅₀ of 0.028 when the control strain reached an OD₅₅₀ of 0.64. Thus the growth of the culture that is being inhibited by a two-day inhibitor will only be 4.4% of that of the control strain at this point, and the inhibitor peptide would have effectively inhibited the growth of the culture by 95.6%. These calculations are consistent with the observation that two-day inhibitors prevent the growth of bacteria on plates for a full 48 hours while the one-day inhibitors only prevent the growth of bacteria on plates for 24 hours.

[0120] All 21 candidates were examined using restriction analysis to determine whether they contained 66 bp inserts as expected. While most of them did, the two day inhibitors pPep3 and pPep14 were found to contain a huge deletion. Sequence analysis of these clones revealed that the deletion had caused the carboxy-terminal end of the inhibitor peptides to become fused to the amino-terminal end of the short 63 amino acid Rop protein. The rop gene which is part of the ColE1 replicon is located downstream from where the oligonucleotide library is inserted into the pLAC11 vector.

[0121] Sequence Analysis of the Top 10 “Anchorless” Inhibitor Peptides. The DNA fragments comprising the sequences encoding the top 10 “anchorless” inhibitor peptides (i.e., excluding the two Rop fusion peptides) were sequenced, and their coding regions are shown in Table 11. Stop codons are represented by stars, and the landmark Bgl II and EcoR I restriction sites for the insert region are underlined. Since the ends of the oligonucleotide from which these inhibitors were constructed contained these restriction sites, the oligonucleotide was not gel isolated when the libraries were prepared in order to maximize the oligonucleotide yields. Because of this, several of the inhibitory clones were found to contain one (n-1) or two (n-2) base deletions in the randomized portion of the oligonucleotide. TABLE 11 Sequence analysis of the insert region from the top 10 inhibitory clones and the peptides that they are predicted to encode pPep1-13 aa  (SEQ ID NO: 34) CAG GAA AGA TCT ATG GTC ACT GAA TTT TGT GGC TTG TTG GAC CAA CTG CCT TAG TAA TAG TGG AAG GCT M V T E F C G L L D Q L P * * * GAA ATT AAT AAG AAT TC (SEQ ID NO: 35) pPep5-16 aa  (SEQ ID NO: 36) CAG GAA AGA TCT ATG TGG CGG GAC TCA TGG ATT AAG GGT AGG GAC GTG GGG TTT ATG GGT TAA AAT AGT M W R D S W I K G R D V G F M G * TTG ATA ATA AGA ATT C (SEQ ID NO: 37) pPep6-42 aa-last 25 aa could form a hydrophobic membrane- spanning domain CAG GAA AGA TCT ATG TCA GGG GGA CAT GTG ACG AGG GAG TGC AAG TCG GCG ATG TCC AAT CGT TGG ATC M S G G H V T R E C K S A M S N R W I TAC GTA ATA AGA ATT CTC ATG TTT GAC AGC TTA TCA TCG ATA AGC TTT AAT GCG GTA GTT TAT CAC AGT Y V I R I L M F D S L S S I S F N A V V Y H S TAA (SEQ ID NO: 38) * (SEQ ID NO: 39) pPep7-6 aa CAG GAA AGA TCT ATG TAT TTG TTC ATC GGA TAA TAC TTA ATG GTC CGC TGG AGA ACT TCA GTT TAA TAA M Y L F I G * (SEQ ID NO: 40) GAA TTC (SEQ ID NO: 41) pPep8-21 a CAG GAA AGA TCT ATG CTT CTA TTT GGG GGG GAC TGG GGG CAG AAA GCC GGA TAC TTT ACT GTG CTA CCG M L L F G G D C G Q K A G Y F T V L P TCA AGG TAA TAA GAA TTC (SEQ ID NO: 42) S R * * (SEQ ID NO: 43) pPep10-20 aa-predicted to be 45% β-sheet -amino acids 6-14 CAG GAA AGA TCT ATG ATT GGG GGA TCG TTG AGC TTC GCG TGG GCA ATA GTT TGT AAT AAG AAT TCT CAT M I G G S L S F A W A I V C N K N S H GTT TGA (SEQ ID NO: 44) V * (SEQ ID NO: 45) pPep12-14 aa  (SEQ ID NO: 46) CAG GAA AGA TCT ATG AAC GGC CGA ACC AAA CGA ATC CGG GAC CCA CCA GCC GCC TAA ACA GCT ACC AGC M N G R T K R I R D P P A A * TGT GGT AAT AAG AAT TC (SEQ ID NO: 47) pPep13-18 aa-predicted to be 72% α-helical-amino acids 3-15 CAG GAA AGA TCT ATG GAC CGT GAA GTG ATG TGT GCG GCA AAA CAG GAA TGG AAG GAA CGA ACG CCA TAG M D R E V M C A A K Q E W K E R T P * (SEQ ID NO: 48) GCC GCG TAA TAA GAA TTC (SEQ ID NO: 49) pPep17-12 aa CAG GAA AGA TCT ATG TAG CCC AAT GCA CTG GGA GCA CGC GTG TTA GGT CTA GAA GCC ACG TAC CCA TTT M * M L G L E A T Y P F AAT CCA TAA TAA GAA TTC (SEQ ID NO: 50) N P * * (SEQ ID NO: 51) pPep19-5 aa CAG GAA AGA TCT ATG AGG GGC GCC AAC TAA GGG GGG GGG AAG GTA TTT GTC CCG TGC ATA ATC TCG GGT M R G A N * (SEQ ID NO: 52) GTT GTC TAA TAA GAA TTC (SEQ ID NO: 53)

[0122] Eight out of the top 10 inhibitors were predicted to encode peptides which terminate before the double TAA TAA termination site which was engineered into the oligonucleotide. Two of the inhibitors, pPep6 and pPep10, which contain deletions within the randomized portion of the oligonucleotide, are terminated beyond the EcoR I site. One of the inhibitors, pPep17, contains a termination signal just after the ATG start codon. However, just downstream from this is a Shine Dalgarno site and a GTG codon which should function as the start codon. Interestingly, the start sites of several proteins such as Rop are identical to that proposed for the pPep17 peptide (G. Cesareni et al., Proc. Natl. Acad. Sci. USA. 79:6313-6317 (1982)). The average and median length for the 8 peptides whose termination signals occurred before or at the double TAA TAA termination site was 13 amino acids.

[0123] The characteristics of the predicted coding regions of the inhibitor peptides proved to be quite interesting. Three out of the top 10 peptides, pPep1, pPep13, and pPep17, contained a proline residue as their last (C-terminal) amino acid. Additionally, one of the peptides, pPep12, contained 2 proline residues near the C-terminus, at the n-2 and n-3 positions. Thus there appears to be a bias for the placement of proline residues at or near the end of several of the inhibitory peptides. Secondary structure analysis predicted that 3 out of the 10 peptides contained a known motif that could potentially form a very stable structure. pPep13, a peptide containing a C-terminal proline, is predicted to be 72% α-helical, pPep10 is predicted to be 45% β-sheet, and pPep6 is predicted to form a hydrophobic membrane spanning domain.

[0124] Verifying that the Inhibitory Clones do not Function as Antisense. To rule out the possibility that the bioactivity of the inhibitory clones resulted from their functioning as antisense RNA or DNA (thus hybridizing to host DNA or RNA) rather than by way of the encoded peptides, the insert regions between the Bgl II and EcoR I sites for the top five inhibitors from Table 10 were recloned into the pLAC11 vector using oligonucleotides which converted the ATG start codon to an ACG codon thus abolishing the start site. In all five cases the new constructs were no longer inhibitory (see Table 12), thus confirming that it is the encoded peptides that causes the inhibition and not the DNA or transcribed mRNA. TABLE 12 Antisense test of the top 5 “anchorless” inhibitory peptides from Inhibitory % inhibition versus Antisense % inhibition versus peptide pLAC11 control construct pLAC11 control pPep1 26 pPep1-anti 0 pPep5 23 pPep5-anti 0 pPep12 80 pPep12-anti 0 pPep13 28 pPep13-anti 0 pPep19 29 pPep19-anti 0

[0125] Growth rates for cells containing the induced inhibitors or antisense constructs were determined and then the % inhibition was calculated by comparing these values to the growth rate of cells that contained the induced pCyt-3 vector.

[0126] Discussion

[0127] Use of the tightly regulable pLAC11 expression vector made possible the identification of novel bioactive peptides. The bioactive peptides identified using the system described in this Example inhibit the growth of the host organism (E. coli) on minimal media. Moreover, bioactive peptides thus identified are, by reason of the selection process itself, stable in the host's cellular environment. Peptides that are unstable in the host cell, whether or not bioactive, will be degraded; those that have short half-lives are, as a result, not part of the selectable pool. The selection system thus makes it possible to identify and characterize novel, stable, degradation-resistant bioactive peptides in essentially a single experiment.

[0128] The stability of the inhibitory peptides identified in this Example may be related to the presence of certain shared structural features. For example, three out of the top 10 inhibitory “anchorless” (i.e., non-Rop fusion) peptides contained a proline residue as their last amino acid. According to the genetic code, a randomly generated oligonucleotide such as the one used in this Example has only a 6% chance of encoding a proline at a given position, yet the frequency of a C-terminal proline among the top ten inhibitory peptides is a full 30%. This 5-fold bias in favor of a C-terminal proline is quite surprising, because although the presence of proline in a polypeptide chain generally protects biologically active proteins against nonspecific enzymatic degradation, a group of enzymes exists that specifically recognize proline at or near the N- and C-termini of peptide substrates. Indeed, proline-specific peptidases have been discovered that cover practically all situations where a proline residue might occur in a potential substrate (D. F. Cunningham et al., Biochimica et Biophysica Acta 1343:160-186 (1997)). For example, although the N-terminal sequences Xaa-Pro-Yaa- and Xaa-Pro-Pro-Yaa (SEQ ID NO: 54) have been identified as being protective against nonspecific N-terminal degradation, the former sequence is cleaved by aminopeptidase P (at the Xaa-Pro bond) and dipeptidyl peptidases IV and II (at the -Pro-Yaa-bond) ) (Table 5, G. Vanhoof et al., FASEB J. 9:73644 (1995); D. F. Cunningham et al., Biochimica et Biophysica Acta 1343:160-186 (1997)); and the latter sequence, present in bradykinin, interleukin 6, factor XII and erythropoietin, is possibly cleaved by consecutive action of aminopeptidase P and dipeptidyl peptidase IV (DPPIV), or by prolyl oligopeptidase (post Pro-Pro bond) (Table 5, G. Vanhoof et al., FASEB J. 9:736-44 (1995)). Prolyl oligopeptidase is also known to cleave Pro-Xaa bonds in peptides that contain an N-terminal acyl-Yaa-Pro-Xaa sequence (D. F. Cunningham et al., Biochimica et Biophysica Acta 1343:160-186 (1997)). Other proline specific peptidases acting on the N-terminus of substrates include prolidase, proline iminopeptidase and prolinase. Prolyl carboxypeptidase and carboxypeptidase P, on the other hand, cleave C-terminal residues from peptides with proline being the preferred P₁ residue (D. F. Cunningham et al., Biochimica et Biophysica Acta 1343:160-186 (1997).

[0129] Also of interest with respect to the stability of the inhibitory peptides, three of the top ten (30%) contained motifs that were predicted, using standard protein structure prediction algorithms, to form stable secondary structures. One of the peptides (which also has a C-terminal proline) was predicted to be 72% α-helical. Another was predicted to be 45% β-sheet; this peptide may dimerize in order to effect the hydrogen bonding necessary to form the β-sheet. A third was predicted to possess a hydrophobic membrane spanning domain. According to these algorithms (see, e.g., P. Chou et al., Adv. Enzymol. 47:45-148 (1978); J. Gamier et al., J. Mol. Biol. 120:97-120 (1978); P. Chou, “Prediction of protein structural classes from amino acid composition,” In Prediction of Protein Structure and the Principles of Protein Conformation (Fasman, G. D. ed.). Plenum Press, New York, N.Y. 549-586 (1990); P. Klein et al., Biochim. Biophys. Acta 815:468-476 (1985)), a randomly-generated oligonucleotide such as the one used in our studies would have had no better than a 1 in a 1000 chance of generating the motifs that occurred in these peptides.

[0130] Finally, two of the three two-day inhibitors proved to be fusion peptides in which the carboxyl terminus of the peptides was fused to the amino terminus of the Rop protein. Rop is a small 63 amino acid protein that consists of two antiparallel α-helices connected by a sharp hairpin loop. It is a dispensable part of the ColE1 replicon which is used by plasmids such as pBr322, and it can be deleted without causing any ill-effects on the replication, partitioning, or copy numbers of plasmids that contain a ColE1 ori (X. Soberon, Gene. 9: 287-305 (1980). Rop is known to possess a highly stable structure (W. Eberle et al., Biochem. 29:7402-7407 (1990); S. Betz et al., Biochemistry 36:2450-2458 (1997)), and thus it could be serving as a stable protein anchor for these two peptides.

[0131] Table 13 lists naturally occurring bioactive peptides whose structures have been determined. Most of these peptides contain ordered structures, further highlighting the importance of structural stabilization. Research on developing novel synthetic inhibitory peptides for use as potential therapeutic agents over the last few years has shown that peptide stability is a major problem that must be solved if designer synthetic peptides are to become a mainstay in the pharmaceutical industry (J. Bai et al., Crit. Rev. Ther. Drug. 12:339-371 (1995); R. Egleton Peptides. 18:1431-1439 (1997); L. Wearley, Crit Rev Ther Drug Carrier Syst. 8: 331-394 (1991). The system described in this Example represents a major advance in the art of peptide drug development by biasing the selection process in favor of bioactive peptides that exhibit a high degree of stability in an intracellular environment. TABLE 13 Structural motifs observed in naturally occurring bioactive peptides Size in Bioactive Amino Peptide acids Structural Motif Reference Dermaseptin 34 α-helix 34 Endorphin 30 α-helix 7 Glucagon 29 α-helix 6 Magainins ^(a) 23 α-helix 5 Mastoparan 14 α-helix 11 Melittin 26 α-helix 44 Motilin 22 α-helix 25 PKI (5-24) 20 α-helix 38 Secretin 27 α-helix 8 Atrial 28 disulfide bonds 33 Natriuretic Peptide Calcitonin 32 disulfide bonds 4 Conotoxins ^(a) 10-30 disulfide bonds 37 Defensins ^(a) 29-34 disulfide bonds 30 EETI II 29 disulfide bonds 23 Oxytocin 9 disulfide bonds 45 Somatostatin 14 disulfide bonds 35 Vasopressin 9 disulfide bonds 20 Bombesin 14 disordered 12 Histatin 24 disordered 51 Substance P 11 disordered 50

Example III Directed Synthesis of Stable Synthetically Engineered Inhibitor Peptides

[0132] These experiments were directed toward increasing the number of bioactive peptides produced by the selection method described in Example II. In the initial experiment, randomized peptides fused to the Rop protein, at either the N- or C- terminus, were evaluated. In the second experiment, nucleic acid sequences encoding peptides containing a randomized internal amino acid sequence flanked by terminal prolines were evaluated. Other experiments included engineering into the peptides an α-helical structural motif, and engineering in a cluster of opposite charges at the N- and C-termini of the peptide.

[0133] Materials and Methods

[0134] Media. Rich LB and minimal M9 media used in this study was prepared as described by Miller (see Example I). Ampicillin was used in rich media at a final concentration of 100 ug/ml and in minimal media at a final concentration of 50 ug/ml. IPTG was added to media at a final concentration of 1 mM.

[0135] Chemicals and Reagents. Extension reactions were carried out using Klenow from New England Biolabs (Bedford, Mass.) while ligation reactions were performed using T4 DNA ligase from Life Sciences (Gaithersburg, Md.) Alkaline phosphatase (calf intestinal mucosa) from Pharmacia (Piscataway, N.J.) was used for dephosphorylation. IPTG was obtained from Diagnostic Chemicals Limited (Oxford, Conn.).

[0136] Bacterial Strains and Plasmids. ALS225, which is MC1061/F′lacIq¹Z+Y+A+, was the E. coli bacterial strain used in this study (see Example I). The genotype for MC1061 is araD139 Δ(araABOIC-leu)7679 Δ(lac)X74 galU galK rpsL hsr− hsm+ as previously described. pLAC11 (Example I), a highly regulable expression vector, was used to make p-Rop(C) and p(N)Rop-fusion vectors as well as the other randomized peptide libraries which are described below.

[0137] Construction of the p-Rop(C) Fusion Vector. The forward primer 5′ TAC TAT AGA TCT ATG ACC AAA CAG GAA AAA ACC GCC 3′ (SEQ ID NO: 55) and the reverse primer 5′ TAT ACG TAT TCA GTT GCT CAC ATG TTC TTT CCT GCG 3′ (SEQ ID NO: 56) were used to PCR amplify a 558 bp DNA fragment using pBR322 as a template. This fragment contained a Bgl II restriction site which was incorporated into the forward primer followed by an ATG start codon and the Rop coding region. The fragment extended beyond the Rop stop codon through the Afl III restriction site in pBR322. The amplified dsDNA was gel isolated, restricted with Bgl II and Afl III, and then ligated into the pLAC11 expression vector which had been digested with the same two restriction enzymes. The resulting p-Rop(C) fusion vector is 2623 bp in size (FIG. 7).

[0138] Construction of the p(N)Rop-Fusion Vector. The forward primer 5′ AAT TCA TAC TAT AGA TCT ATG ACC AAA CAG GAA AAA ACC GC 3′ (SEQ ID NO: 57) and the reverse primer 5′ TAT ATA ATA CAT GTC AGA ATT CGA GGT TTT CAC CGT CAT CAC 3′ (SEQ ID NO: 58) were used to PCR amplify a 201 bp DNA fragment using pBR322 as a template. This fragment contained a Bgl II restriction site which was incorporated into the forward primer followed by an ATG start codon and the Rop coding region. The reverse primer placed an EcoR I restriction site just before the Rop TGA stop codon and an Afl III restriction site immediately after the Rop TGA.stop codon. The amplified dsDNA was gel isolated, restricted with Bgl II and Afl III, and then ligated into the pLAC11 expression vector which had been digested with the same two restriction enzymes. The resulting p(N)Rop-fusion vector is 2262 bp in size (FIG. 8).

[0139] Generation of Rop Fusion Randomized Peptide Libraries. Peptide libraries were constructed as described in Example II. The synthetic oligonucleotide 5′ TAC TAT AGA TCT ATG NN)₂₀ CAT AGA TCT GCG TGC TGT GAT 3′ (SEQ ID NO: 59) was used to construct the randomized peptide libraries for use with the p-Rop(C) fusion vector, substantially as described in Example II. The complementary strand of this oligonucleotide was generated by a fill-in reaction with Klenow using an equimolar amount of the oligonucleotide primer 5′ ATC ACA GCA CGC AGA TCT ATG 3′ were used (SEQ ID NO: 60). After extension the resulting dsDNA was digested with Bgl II and ligated into the pLAC11 expression vector which had been digested with the same restriction enzyme and subsequently dephosphorylated using alkaline phosphatase. Because of the way the oligonucleotide library has been engineered, either orientation of the incoming digested double-stranded DNA fragment results in a fusion product.

[0140] To construct the randomized peptide libraries for use with the p(N)Rop-fusion vector, the randomized oligonucleotide 5′ TAC TAT GAA TTC (NNN)₂₀ GAA TTC TGC CAC CAC TAC TAT 3′ (SEQ ID NO: 61), and the primer 5′ ATA GTA GTG GTG GCA GAA TTC 3′ (SEQ ID NO: 62) were used. After extension the resulting dsDNA was digested with EcoRI and ligated into the pLAC11 expression vector which had been digested with the same restriction enzyme and subsequently dephosphorylated using alkaline phosphatase. Because of the way the oligonucleotide library has been engineered, either orientation of the incoming digested double-stranded DNA fragment results in a fusion product.

[0141] Generation of a Randomized Peptide Library Containing Terminal Prolines. Randomized 20 amino acid peptide libraries containing two proline residues at both the amino and the carboxy terminal ends of the peptides were constructed using the synthetic oligonucleotide 5′ TAC TAT AGA TCT ATG CCG CCG (NNN)16 CCG CCG TAA TAA GAA TTC GTA CAT 3′ (SEQ ID NO: 63). The complementary strand of the 93 base randomized oligonucleotide was generated by filling-in with Klenow using the oligonucleotide primer 5′ ATG TAC GAA TTC TTA TTA CGG CGG 3′ (SEQ ID NO: 64). After extension the resulting dsDNA was digested with Bgl II and EcoR I and ligated into the pLAC11 expression vector which had been digested with the same two restriction enzymes. Because the initiating methionine of the peptides coded by this library is followed by a proline residue, the initiating methionine will be removed (F. Sherman et al, Bioessays 3: 27-31 (1985)). Thus the peptide libraries encoded by this scheme are 20 amino acids in length.

[0142] Generation of a Randomized Hydrophilic α-Helical Peptide Library. Table 14 shows the genetic code highlighted to indicate certain amino acid properties. TABLE 14 Genetic Code Highlighted to Indicate Amino Acid Properties TTT phe h_(a) TCT ser TAT tyr b_(a) TGT CYS TTC phe h_(a) TCC ser TAC tyr ba TGC cys TTA leu H_(a) TCA ser TAA OCH TGA OPA TTG leu H_(a) TCG ser TAG AMB TGG trp CTT leu H_(a) CCT pro B_(a) CAT his h_(a) CGT arg CTC leu H_(a) CCC pro B_(a) CAC his h_(a) CGC arg CTA leu H_(a) CCA pro B_(a) CAA gln h_(a) CGA arg CTG leu H_(a) CCG pro B_(a) CAG gln h_(a) CGG arg ATT ile h_(a) ACT thr AAT asn b_(a) AGT ser ATC ile h_(a) ACC thr AAC asn b_(a) AGC ser ATA ile h_(a) ACA thr AAA lys h_(a) AGA arg ATG met H_(a) ACG thr AAG lys h_(a) AGG arg GTT val h_(a) GCT ala H_(a) GAT asp h_(a) GGT gly B_(a) GTC val h_(a) GCC ala H_(a) GAC asp h_(a) GGC gly B_(a) GTA val h_(a) GCA ala H_(a) GAA glu H_(a) GGA gly B_(a) GTG val h_(a) GCG ala H_(a) GAG glu H_(a) GGG gly B_(a) # assignments given in this table are the consensus agreement from serveral different sources. Hydrophilic versus hydrophobic assignments for the amino acids were made from data found in Wolfenden et. al. (Biochemistry. 20:849-55 (1981)); Miller et. al. (J. Mol. Biol. 196:641-656 (1987)); and Roseman (J. Mol. Biol. 200:513-22(1988)). The propensity for amino acids to form α-helical structures were obtained from consensus agreements of the Chou and Fasman (P. Chou et al., # Adv. Enzymol. 47:45-148 (1978); P. Chou, “Prediction of protein structural classes from amino acid compositions,” in Prediction of protein structure and the principles of protein conformation (G. Fasman, G.D. ed.). Plenum Press, New York, N.Y. 549-586 (1990)); Garnier, Osguthorpe, and Robson (J. Mol. Biol. 120:97-120 (1978)); and O'Neill and DeGrado (Science. 250:646-651 (1990)) methods for predicting secondary structure.

[0143] By analyzing the distribution pattern of single nucleotides in the genetic code relative to the properties of the amino acids encoded by each nucleotide triplet, a novel synthetic approach was identified that would yield randomized 18 amino acid hydrophilic peptide libraries with a propensity to form α-helices. According to Table 14, the use of a [(CAG)A(TCAG)] codon mixture yields the hydrophilic amino acids His, Gln, Asn, Lys, Asp, and Glu. These amino acids are most often associated with α-helical motifs except for asparagine, which is classified as a weak α-helical breaker. If this codon mixture was used to build an α-helical peptide, asparagine would be expected to occur in about 17% of the positions, which is acceptable in an α-helical structure according to the secondary structure prediction rules of either Chou and Fasman (P. Chou et al., Adv. Enzymol. 47:45-148 (1978); P. Chou, “Prediction of protein structural classes from amino acid compositions,” in Prediction of protein structure and the principles of protein conformation (G. Fasman, G. D. ed.). Plenum Press, New York, N.Y. 549-586 (1990)) or Gamier, Osguthorpe, and Robson (J. Gamier et al., J Mol. Biol. 120:97-120 (1978)). Additionally, several well-characterized proteins have been observed to contain up to three b_(a) breaker amino acids within a similarly sized α-helical region of the protein (T. Creighton, “Conformational properties of polypeptide chains,” in Proteins: structures and molecular properties, W.H. Freeman and Company, N.Y., 182-186 (1993)). Since in most a-helices there are 3.6 amino acids per complete turn, the 18 amino acid length was chosen in order to generate α-helical peptides which contained 5 complete turns. Moreover, the use of hydrophilic amino acids would be expected to yield peptides which are soluble in the cellular cytosol.

[0144] Randomized 18 amino acid hydrophilic α-helical peptide libraries were synthesized using the synthetic oligonucleotide 5′ TAC TAT AGA TCT ATG (VAN)₁₇ TAA TAA GAA TTC TGC CAG CAC TAT 3′ (SEQ ID NO: 65). The complementary strand of the 90 base randomized oligonucleotide was generated by filling-in with Klenow using the oligonucleotide primer 5′ ATA GTG CTG GCA GAA TTC TTA TTA 3′ (SEQ ID NO: 66). After extension the resulting dsDNA was digested with Bgl II and EcoR I and ligated into the pLAC11 expression vector which had been digested with the same two restriction enzymes.

[0145] Generating a Randomized Peptide Library Containing the ±Charge Ending Motif. Randomized peptide libraries stabilized by the interaction of oppositely charge amino acids at the amino and carboxy termini were generated according to the scheme shown in FIG. 9. To maximize the potential interactions of the charged amino acids, the larger acidic amino acid glutamate was paired with the smaller basic amino acid lysine, while the smaller acidic amino acid aspartate was paired with the larger basic amino acid arginine. To construct the randomized peptide libraries, the synthetic oligonucleotide 5′ TAC TAT AGA TCT ATG GAA GAC GAA GAC (NNN)₁₆ CGT AAA CGT AAA TAA TAA GAA TTC GTA CAT 3′ (SEQ ID NO: 67) and the oligonucleotide primer 5′ ATG TAC GAA TTC TTA TTA TTT ACG TTT ACG 3′ (SEQ ID NO: 68) were used. After extension the resulting dsDNA was digested with Bgl II and EcoR I and ligated into the pLAC11 expression vector which had been digested with the same two restriction enzymes.

[0146] For all libraries of randomized oligonucleotides, N denotes that an equimolar mixture of the four nucleotides A, C, G, and T was used, and V denotes that an equimolar mixture of the three nucleotides A, C and G was used. The resulting libraries were transformed into electrocompetent ALS225 E. coli cells (Example I) under repressed conditions as described in Example II.

[0147] Screening of Transformants to Identify Inhibitor Clones. Transformants were initially screened using the grid-patching technique to identify any that could not grow on minimal media as described in Example II when the peptides were overproduced. To verify that all the inhibitors were legitimate, plasmid DNA was made from each inhibitory clone, transformed into a fresh background, then checked to make sure that they were still inhibitory on plates and that their inhibition was dependent on the presence of the inducer, IPTG, as in Example II.

[0148] Growth Rate Analysis in Liquid Media. Inhibition strength of the peptides was assessed by subjecting the inhibitory clones-to a growth rate analysis in liquid media. Minimal or rich cultures containing either the inhibitor to be tested or the relevant vector as a control were diluted to an initial OD₅₅₀ of approximately 0.01 using new media and induced with 1 mM IPTG. OD₅₅₀ readings were then taken hourly until the cultures had passed log phase. Growth rates were determined as the spectrophotometric change in OD₅₅₀ per unit time within the log phase of growth, and inhibition of the growth rate was calculated for the inhibitors using the appropriate vector as a control.

[0149] Results

[0150] Isolation and Characterization of Inhibitor Peptides that are Fused at Their Carboxy Terminal End to the Amino Terminal End of the Rop Protein. Approximately 10,000 peptides protected by the Rop protein at their carboxy terminal end were screened using the grid-patching technique described in Example II, and 16 two day inhibitors were isolated. The inhibitory effects were determined as described in the Example II, using pRop(C) as a control. Unlike the anchorless inhibitors identified in Example II that were only inhibitory on minimal media, many of the Rop fusion inhibitors were also inhibitory on rich media as well, which reflects increased potency. As indicated in Table 15, the inhibitors inhibited the bacterial growth rate at levels that averaged 90% in minimal media and at levels that averaged 50% in rich media. The data in Table is the average of duplicate experiments. TABLE 15 Inhibitory effects of peptide inhibitors stabilized by fusing the carboxy terminal end of the peptide to the amino terminal end of the Rop protein (Rop(C) fusion peptide inhibitors % inhibition in % inhibition in Inhibitor minimal media rich media PRop(C)1 87 47 PRop(C)2 99 58 PRop(C)3 85 54 PRop(C)4 98 49 PRop(C)5 95 54 PRop(C)6 99 46 PRop(C)7 91 59 PRop(C)8 86 51 PRop(C)9 93 57 PRop(C)10 91 35

[0151] Isolation and Characterization of Inhibitor Peptides that are Fused at Their Amino Terminal End to the Carboxy Terminal End of the Rop Protein. Approximately 6000 peptides protected at their amino terminal end by Rop protein were screened using the grid-patching technique described in Example II, and 14 two day inhibitors were isolated. As observed for the Rop fusion peptides isolated using the p-Rop(C) vector, most of the inhibitor peptides isolated using the p(N)Rop-vector were inhibitory on rich media as well as minimal media. The inhibitors were verified as described hereinabove and subjected to growth rate analysis using p(N)Rop- as a control in order to determine their potency. As indicated in Table 16, the inhibitors inhibited the bacterial growth rate at levels that averaged 90% in minimal media and at levels that averaged 40% in rich media. The data in Table 16 is the average of duplicate experiments. TABLE 16 Inhibitory effects of peptide inhibitors stabilized by fusing the amino terminal end of the peptide to the carboxy terminal end of the Rop protein (Rop(N) fusion peptide inhibitors) % inhibition in % inhibition in Inhibitor minimal media rich media pRop(N)1 81 30 pRop(N)2 96 53 pRop(N)3 95 43 pRop(N)4 92 38 pRop(N)5 99 33 pRop(N)6 93 38 pRop(N)7 87 34 pRop(N)8 91 44 pRop(N)9 95 37 pRop(N)10 96 40

[0152] Isolation and Characterization of Anchorless Inhibitor Peptides Containing Two Prolines at Both Their Amino Terminal and Carboxy Terminal Ends. Approximately 7500 peptides were screened using the grid-patching technique described in Example II, and 12 two day inhibitors were isolated. As indicated in Table 17, the top ten inhibitors inhibited the bacterial growth rate at levels that averaged 50% in minimal media. The inhibitory effects were determined as described in the text using pLAC11 as a control. The data in Table 17 is the average of duplicate experiments. TABLE 17 Inhibitory effects of peptide inhibitors stabilized by two proline residues at both the amino and carboxy terminal ends of the peptide % inhibition in Inhibitor minimal media pPro1 50 pPro2 49 pPro3 50 pPro4 59 pPro5 52 pPro6 93 pPro7 54 pPro8 42 pPro9 41 pPro1O 42

[0153] Sequence analysis of the coding regions for the top ten inhibitors is shown in Table 19. The landmark Bgl II and EcoR I restriction sites for the insert region are underlined, as are the proline residues.

[0154] Since the ends of the oligonucleotide from which these inhibitors were constructed contained Bgl II and EcoR I restriction sites, the oligonucleotide was not gel isolated when the libraries were prepared in order to maximize the oligonucleotide yields. Because of this, three of the inhibitory clones, pPro2, Ppro5, and pPro6 were found to contain deletions in the randomized portion of the oligonucleotide. TABLE 18 Sequence analysis of the insert region from the proline peptides pPro1-21aa AGA TCT ATG CCG CCG ATT CTA TGG GGC GAA GCG AGA AAG CGC TTG TGG GGT GGG GAT CAT ACA CCG CCG TAA TAA M P P I L W G E A R K R L W G G D H T P P * * (SEQ ID NO: 70) GAA TTC (SEQ ID NO: 69) pPro2-27aa AGA TCT ATG CCG CCG CCG TTG GAT ATT GTG TCG GGT ATT GAG GTA GGG GGG CAT TTG TGG TGC CGC CGT ATT AAG M P P P L D I V S G I E V G G H L W C R R I K AAT TCT CAT GTT TGA (SEQ ID NO: 71) N S H V * (SEQ ID NO: 72) pPro3-8aa AGA TCT ATG CCG CCG GAC AAT CCG GTC CTG TGA TGA AGC GGA GGT CGA CCA AGG GGA TAT CAG CCG CCG TAA TAA M P P D N P V L * * (SEQ ID NO: 74) GAA TTC (SEQ ID NO: 73) pPro4-9aa AGA TCT ATG CCG CCG CTA TTG GAC GGA GAT GAC AAA TAG ATA TAT GCG TGG TTG TTT TTC TGT CCG CCG TAA TAA M P P L L D G D D K * (SEQ ID NO: 76) GAA TTC (SEQ ID NO: 75) pPro5-10aa AGA TCT ATG CCG CCG AGG TGG AAG ATG TTG ATA AGA CAG TGA CAG ATG CGT TCC ATT ACT CCC GCC GTA ATA AGA M P P R W K M L I R Q * (SEQ ID NO: 78) ATT C (SEQ ID NO: 77) pPro6-7aa AGA TCT ATG ATG AGA GTA GCG CCG CCG TAA TAA GAA TTC (SEQ ID NO: 79) M M R V A P P * * (SEQ ID NO: 80) pPro7-14aa AGA TCT ATG CCG CCG TTG CGC GGG GCA TGC GAT GTA TAT GGG GTA AAT TGA ATG TCT TGT GGG CCG CCG TAA TAA M P P L R G A C D V Y G V N * (SEQ ID NO: 82) GAA TTC (SEQ ID NO: 81) pPro8-21aa AGA TCT ATG CCG CCG GGG AGA GGG GAA GCG GTG GGA GTG ACA TGC TTG AGC GCG AAC GTG TAC CCG CCG TAA TAA M P P G R G E A V G V T C L S A N V Y P P * * (SEQ ID NO: 84) GAA TTC (SEQ ID NO: 83) pPro9-21aa AGA TCT ATG CCG CCG GGA AGG GTA GTG TTC TTT GTC GCT ATC TTT GTT TCC GCA ATA TGC CTC CCG CCG TAA TAA M P P G R V V F F V A I F V S A I C L P P * * (SEQ ID NO: 86) GAA TTC (SEQ ID NO: 85) pPro10-21aa AGA TCT ATG CCG CCG AGG TTC GCT CAT GAG AGT GTT AAA GGG CTG GGG GAC GTT ACA AAA GCT CCG CCG TAA TAA M P P R F A H E S V K G L G D V T K A P P * * (SEQ ID NO: 88) GAA TTC (SEQ ID NO: 87)

[0155] All the inhibitors were found to contain two proline residues at either their amino or carboxy ternini as expected. Four inhibitors contained two proline residues at both their amino and carboxy termini, five inhibitors contained two proline residues at only their amino ternini, and one inhibitor contained two proline residues at only its carboxy terminus.

[0156] Isolation and Characterization of Anchorless Hydrophilic Inhibitor Peptides Stabilized by an α-Helical Motif. Approximately 12,000 peptides were screened using the grid-patching technique and 5 two day inhibitors were isolated. The inhibitors were verified as already described for the Rop-peptide fusion studies and subjected to growth rate analysis using pLAC11 as a control in order to determine their potency. As indicated in Table 19, the inhibitor peptides inhibited the bacterial growth rate at levels that averaged 50% in minimal media. The averaged values of two independent determinations are shown. TABLE 19 Inhibitory effects of the hydrophilic α-helical peptides % inhibition in Inhibitor minimal media pHelix1 67 pHelix2 46 pHelix3 48 pHelix4 45 pHelix5 42

[0157] Sequence analysis of the coding regions for the 5 inhibitors is shown in Table 20. The landmark Bgl II and EcoR I restriction sites for the insert region are underlined. Since the ends of the oligonucleotide from which these inhibitors were constructed contained these restriction sites, the oligonucleotide was not gel isolated when the libraries were prepared in order to maximize the oligonucleotide yields. Because of this, two of the inhibitory clones, pHelix2 and pHelix3, were found to contain deletions in the randomized portion of the oligonucleotide. The predicted α-helical content of these peptides is indicated in Table 20 according to the secondary structure prediction rules of Garnier, Osguthorpe, and Robson (J. Garnier et al., J. Mol. Biol. 120:97-120 (1978)) prediction rules. TABLE 20 Sequence analysis of the insert region from the hydrophilic α-helical peptides pHelix1-18aa, 83% α-helical (SEQ ID NO: 89) AGA TCT ATG CAT GAC GAA CAA GAG GAG GAG CAC AAT AAA AAG GAT AAC GAA AAA GAA CAC TAA TAA GAA M H D E Q E E E H N K K D N E K E H TTC  SEQ ID NO: 90 pHelix2-22aa, 68% α-helical AGA TCT ATG CAG CAG GAG CAC GAG CAA GGC AGG ATG AGC AAG AGG ATG AAG AAT AAT AAG AAT TCT CAT M Q Q E H E Q G R M S K R M K N N K N S H GTT TGA (SEQ ID NO: 91) V * (SEQ ID NO: 92) pHelix3-22aa, 55% α-helical AGA TCT ATG AAC CAT CAT AAT GAG GCC ATG ATC AAC ACA ATG AAA ACG AGG AAT AAT AAG AAT TCT CAT M N H H N E A M I N T M K T R N N K N S H GTT TGA (SEQ ID NO: 93) V * (SEQ ID NO: 94) pHelix4-18aa, 17% α-helical  (SEQ ID NO: 96) AGA TCT ATG AAC GAC GAC AAT CAG CAA GAG GAT AAT CAT GAT CAG CAT AAG GAT AAC AAA TAA TAA GAA M N D D N Q Q E D N H D Q H K D N K * TTC (SEQ ID NO: 95) pHelix5-18aa, 50% α-helical  (SEQ ID NO: 98) AGA TCT ATG CAA GAG CAG GAT CAG CAT AAT GAT AAC CAT CAC GAG GAT AAA CAT AAG AAG TAA TAA GAA M Q E Q D Q H N D N H H E D K H K K * * (SEQ ID NO: 98) TTC (SEQ ID NO: 97)

[0158] According to Garnier, Osguthorpe, and Robson secondary structure prediction, all of the encoded peptides are expected to be largely α-helical except for pHelix4. Interestingly, pHelix1 which had the highest degree of α-helical content was also the most potent inhibitory peptide that was isolated in this study.

[0159] Isolation and Characterization of Anchorless Inhibitor Peptides Stabilized by an Opposite Charge Ending Motif. Approximately 20,000 peptides were screened using the grid-patching technique and 6 two day inhibitors were isolated. The inhibitors were verified as already described for the Rop-peptide fusion studies and subjected to growth rate analysis using pLAC11 as a control in order to determine their potency. As indicated in Table 21, the inhibitor peptides inhibited the bacterial growth rate at levels that averaged 50% in minimal media. The averaged values of two independent determinations are shown. TABLE 21 Inhibitory effects of peptide inhibitors that are stabilized by the opposite charge ending motif % inhibition in Inhibitor minimal media p +/− 1 41 p +/− 2 43 p +/− 3 48 p +/− 4 60 p +/− 5 54 p +/− 6 85

[0160] Sequence analysis of the coding regions for the six inhibitors is shown in Table 22. The landmark Bgl II and EcoR I restriction sites for the insert region are underlined. With the exception of p±4 which was terminated prematurely, the coding regions for the inhibitors were as expected based on the motif that was used to generate the peptide libraries. TABLE 22 Sequence analysis of the insert region from the opposite charge ending peptides p+/−1-25aa AGA TCT ATG GAA GAC GAA GAC GAG GGT GCG TCA GCG TGG GGA GCA GAA CTT TGG TCG TGG CAG TCG GTG M E D E D E G A S A W G A E L W S W Q S V CGT AAA CGT AAA TAA TAA GAA TTC (SEQ ID NO: 99) R K R K * * (SEQ ID NO: 100) p+/−2-25aa AGA TCT ATG GAA GAC GAA GAC GGT CTA GGC ATG GGG GGT GGG TTG GTC AGG CTC ACT TTA TTA TTC TTC M E D E D G L G M G G G L V R L T L L F F CGT AAA CGT AAA TAA TAA GAA TTC (SEQ ID NO: 101) R K R K * * (SEQ ID NO: 102) p+/−3-25aa AGA TCT ATG GAA GAC GAA GAC GGG GAG AGG ATC CAG GGG GCC CGC TGT CCA GTA GCG CTG GTA GAT AGA M E D E D G E R I Q G A R C P V A L V D R CGT AAA CGT AAA TAA TAA GAA TTC (SEQ ID NO: 103) R K R K * * (SEQ ID NO: 104) p+/−4-11aa AGA TCT ATG GAA GAC GAA GAC GAC AGG GGG CGT GGG CGG TAG CTT TAA GTT GCG CTA AGT TGC GAG ATA M E D E D D R G R G R * (SEQ ID NO: 105) CGT AAA CGT AAA TAA TAA GAA TTC (SEQ ID NO: 106) p+/−5-25aa AGA TCT ATG GAA GAC GAA GAC GGG GGG GCC GGG AGG AGG GCC TGT CTT TGT TCC GCG CTT GTT GGG GAA M E D E D G G A G R R A C L C S A L V G E CGT AAA CGT AAA TAA TAA GAA TTC (SEQ ID NO: 107) R K R K * * (SEQ ID NO: 108) p+/−6-25aa AGA TCT ATG GAA GAC GAA GAC AAG CGT CGC GAG AGG AGT GCA AAA GGG CGT CAT GTC GGT CGG TCG ATG M E D E D K R R E R S A K G R H V G R S M CGT AAA CGT AAA TAA GAC TGT (SEQ ID NO: 109) R K R K * (SEQ ID NO: 110)

[0161] Discussion

[0162] In Example II, where fully randomized peptides were screened for inhibitory effect, only three peptides (one “anchorless” and two unanticipated Rop fusions resulting from deletion) were identified out of 20,000 potential candidates as a potent (i.e., two day) inhibitor of E. coli bacteria. Using a biased synthesis as in this Example, it was possible to significantly increase the frequency of isolating potent growth inhibitors (see Table 23). TABLE 23 Summary of the frequency at which the different types of inhibitor peptides can be isolated Frequency at Type of inhibitor which a two day inhibitor peptide peptide can be isolated Reference anchorless 1 in 20,000 Example II protected at the 1 in 625 This example C-terminal end via Rop protected at the 1 in 429 This example N-terminal end via Rop protected at both the 1 in 625 This example C-terminal and N-terminal end via two prolines protected with an 1 in 2,400 This example α-helix structural motif protected with an 1 in 3,333 This example opposite charge ending motif

[0163] Many more aminopeptidases have been identified than carboxypeptidases in both prokaryotic and eukaryotic cells (J. Bai, et al., Pharm. Res. 9:969-978 (1992); J. Brownlees et al., J. Neurochem. 60:793-803 (1993); C. Miller, In Escherichia coli and Salmonella typhimurium cellular and molecular biology, 2nd edition (Neidhardt, F. C. ed.), ASM Press, Washington, D.C. 1:938-954 (1996)). In the Rop fusion studies, it might have therefore been expected that stabilizing the amino terminal end of the peptide would have been more effective at preventing the action of exopeptidases than stabilizing the carboxy end of the peptides. Surprisingly, it was found that stabilizing either end of the peptide caused about the same effect.

[0164] Peptides could also be stabilized by the addition of two proline residues at the amino and/or carboxy termini, the incorporation of opposite charge ending amino acids at the amino and carboxy termini, or the use of helix-generating hydrophilic amino acids. As shown in Table 23, the frequency at which potent inhibitor peptides could be isolated increased significantly over that of the anchorless peptides characterized in Example II.

[0165] These findings can be directly implemented to design more effective peptide drugs that are resistant to degradation by peptidases. In this example, several strategies were shown to stabilize peptides in a bacterial host. Because the aminopeptidases and carboxypeptidases that have been characterized in prokaryotic and eukaryotic systems appear to function quite similarly (C. Miller, In Escherichia coli and Salmonella typhimurium cellular and molecular biology, 2nd edition (Neidhardt, F. C. ed.), ASM Press, Washington, D.C. 1:938-954 (1996); N. Rawlings et al., Biochem J. 290:205-218 (1993)), the incorporation of on or more of these motifs into new or known peptide drugs should slow or prevent the action of exopeptidases in a eukaryotic host cell as well.

Sequence Listing Free Text

[0166] SEQ ID NO:2

[0167] peptide sequence having opposite charge ending motif

[0168] SEQ ID NOs:3-4

[0169] stabilized angiotensin

[0170] SEQ ID NOs:6-19, 24-28, 55-58, 60, 62, 64, 66, 68

[0171] primer

[0172] SEQ ID NOs:20-22

[0173] primer fragment

[0174] SEQ ID NOs:23, 59, 61, 63, 65, 67

[0175] randomized oligonucleotide

[0176] SEQ ID NOs:29-33

[0177] antisense oligonucleotide

[0178] SEQ ID NOs:34, 36, 39, 40, 43, 45, 46, 48, 51, 52, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 105, 108, 110

[0179] stabilized peptide

[0180] SEQ ID NOs:35, 37, 38, 41, 42, 44,47, 49, 50, 53, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 106, 107, 109

[0181] nucleic acid encoding stabilized peptide

[0182] SEQ ID NO:54

[0183] N-terminal protective sequence

[0184] The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claim.

1 110 1 133 DNA Escherichia coli 1 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 60 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 120 ggaaacagct atg 133 2 25 PRT Artificial Sequence Description of Artificial Sequence peptide having opposite charge ending motif 2 Met Glu Asp Glu Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Arg Lys Arg Lys 20 25 3 14 PRT Artificial Sequence Description of Artificial Sequence stabilized angiotensin 3 Pro Pro Asp Arg Val Tyr Ile His Pro Phe His Ile Pro Pro 1 5 10 4 18 PRT Artificial Sequence Description of Artificial Sequence stabilized angiotensin 4 Glu Asp Glu Asp Asp Arg Val Tyr Ile His Pro Phe His Ile Arg Lys 1 5 10 15 Arg Lys 5 10 PRT Homo sapiens 5 Asp Arg Val Tyr Ile His Pro Phe His Ile 1 5 10 6 20 DNA Artificial Sequence Description of Artificial Sequence primer 6 gttgccattg ctgcaggcat 20 7 42 DNA Artificial Sequence Description of Artificial Sequence primer 7 attgaattca taagatcttt cctgtgtgaa attgttatcc gc 42 8 37 DNA Artificial Sequence Description of Artificial Sequence primer 8 attgaattca ccatggacac catcgaatgg tgcaaaa 37 9 19 DNA Artificial Sequence Description of Artificial Sequence primer 9 gttgttgcca ttgctgcag 19 10 43 DNA Artificial Sequence Description of Artificial Sequence primer 10 tgtatgaatt cccgggtacc atggttgaag acgaaagggc ctc 43 11 36 DNA Artificial Sequence Description of Artificial Sequence primer 11 tactatagat ctatgaccat gattacggat tcactg 36 12 36 DNA Artificial Sequence Description of Artificial Sequence primer 12 tacataaagc ttggcctgcc cggttattat tatttt 36 13 47 DNA Artificial Sequence Description of Artificial Sequence primer 13 tatcatctgc agaggaaaca gctatgacca tgattacgga ttcactg 47 14 47 DNA Artificial Sequence Description of Artificial Sequence primer 14 tacatactcg agcaggaaag cttggcctgc ccggttatta ttatttt 47 15 47 DNA Artificial Sequence Description of Artificial Sequence primer 15 tatcatggat ccaggaaaca gctatgacca tgattacgga ttcactg 47 16 36 DNA Artificial Sequence Description of Artificial Sequence primer 16 tactatagat ctatggctat cgacgaaaac aaacag 36 17 40 DNA Artificial Sequence Description of Artificial Sequence primer 17 atatataagc ttttaaaaat cttcgttagt ttctgctacg 40 18 35 DNA Artificial Sequence Description of Artificial Sequence primer 18 tactatagat ctatgaacaa aggtgtaatg cgacc 35 19 35 DNA Artificial Sequence Description of Artificial Sequence primer 19 attagtgaat tcgcacaatc tctgcaataa gtcgt 35 20 15 DNA Artificial Sequence Description of Artificial Sequence primer fragment 20 agatcttatg aattc 15 21 15 DNA Artificial Sequence Description of Artificial Sequence primer fragment 21 agatcttatg aattc 15 22 15 DNA Artificial Sequence Description of Artificial Sequence primer fragment 22 agatcttatg aattc 15 23 93 DNA Artificial Sequence Description of Artificial Sequence randomized oligonucleotide 23 tactatagat ctatgnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnntaata agaattctcg aca 93 24 18 DNA Artificial Sequence Description of Artificial Sequence primer 24 tgtcgagaat tcttatta 18 25 20 DNA Artificial Sequence Description of Artificial Sequence primer 25 tcattaatgc agctggcacg 20 26 20 DNA Artificial Sequence Description of Artificial Sequence primer 26 ttcatacacg gtgcctgact 20 27 20 DNA Artificial Sequence Description of Artificial Sequence primer 27 tagctcactc attaggcacc 20 28 20 DNA Artificial Sequence Description of Artificial Sequence primer 28 gatgacgatg agcgcattgt 20 29 92 DNA Artificial Sequence Description of Artificial Sequence antisense oligonucleotide 29 tactatagat ctacggtcac tgaattttgt ggcttgttgg accaactgcc ttagtaatag 60 tggaaggctg aaattaataa gaattctcga ca 92 30 91 DNA Artificial Sequence Description of Artificial Sequence antisense oligonucleotide 30 tactatagat ctacgtggcg ggactcatgg attaagggta gggacgtggg gtttatgggt 60 taaaatagtt tgataataag aattctcgac a 91 31 92 DNA Artificial Sequence Description of Artificial Sequence antisense oligonucleotide 31 tactatagat ctacgaacgg ccgaaccaaa cgaatccggg acccaccagc cgcctaaaca 60 gctaccagct gtggtaataa gaattctcga ca 92 32 93 DNA Artificial Sequence Description of Artificial Sequence antisense oligonucleotide 32 tactatagat ctacggaccg tgaagtgatg tgtgcggcaa aacaggaatg gaaggaacga 60 acgccatagg ccgcgtaata agaattctcg aca 93 33 93 DNA Artificial Sequence Description of Artificial Sequence antisense oligonucleotide 33 tactatagat ctacgagggg cgccaactaa ggggggggga aggtatttgt cccgtgcata 60 atctcgggtg ttgtctaata agaattctcg aca 93 34 13 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 34 Met Val Thr Glu Phe Cys Gly Leu Leu Asp Gln Leu Pro 1 5 10 35 86 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 35 caggaaagat ctatggtcac tgaattttgt ggcttgttgg accaactgcc ttagtaatag 60 tggaaggctg aaattaataa gaattc 86 36 16 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 36 Met Trp Arg Asp Ser Trp Ile Lys Gly Arg Asp Val Gly Phe Met Gly 1 5 10 15 37 85 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 37 caggaaagat ctatgtggcg ggactcatgg attaagggta gggacgtggg gtttatgggt 60 taaaatagtt tgataataag aattc 85 38 141 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 38 caggaaagat ctatgtcagg gggacatgtg acgagggagt gcaagtcggc gatgtccaat 60 cgttggatct acgtaataag aattctcatg tttgacagct tatcatcgat aagctttaat 120 gcggtagttt atcacagtta a 141 39 42 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 39 Met Ser Gly Gly His Val Thr Arg Glu Cys Lys Ser Ala Met Ser Asn 1 5 10 15 Arg Trp Ile Tyr Val Ile Arg Ile Leu Met Phe Asp Ser Leu Ser Ser 20 25 30 Ile Ser Phe Asn Ala Val Val Tyr His Ser 35 40 40 6 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 40 Met Tyr Leu Phe Ile Gly 1 5 41 75 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 41 caggaaagat ctatgtattt gttcatcgga taatacttaa tggtccgctg gagaacttca 60 gtttaataag aattc 75 42 87 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 42 caggaaagat ctatgcttct atttgggggg gactgcgggc agaaagccgg atactttact 60 gtgctaccgt caaggtaata agaattc 87 43 20 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 43 Met Leu Leu Phe Gly Gly Asp Cys Gly Lys Ala Gly Tyr Phe Thr Val 1 5 10 15 Leu Pro Ser Arg 20 44 75 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 44 caggaaagat ctatgattgg gggatcgttg agcttcgcct gggcaatagt ttgtaataag 60 aattctcatg tttga 75 45 20 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 45 Met Ile Gly Gly Ser Leu Ser Phe Ala Trp Ala Ile Val Cys Asn Lys 1 5 10 15 Asn Ser His Val 20 46 14 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 46 Met Asn Gly Arg Thr Lys Arg Ile Arg Asp Pro Pro Ala Ala 1 5 10 47 86 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 47 caggaaagat ctatgaacgg ccgaaccaaa cgaatccggg acccaccagc cgcctaaaca 60 gctaccagct gtggtaataa gaattc 86 48 18 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 48 Met Asp Arg Glu Val Met Cys Ala Ala Lys Gln Glu Trp Lys Glu Arg 1 5 10 15 Thr Pro 49 87 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 49 caggaaagat ctatggaccg tgaagtgatg tgtgcggcaa aacaggaatg gaaggaacga 60 acgccatagg ccgcgtaata agaattc 87 50 87 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 50 caggaaagat ctatgtagcc caatgcactg ggagcacgcg tgttaggtct agaagccacg 60 tacccattta atccataata agaattc 87 51 12 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 51 Met Leu Gly Leu Glu Ala Thr Tyr Pro Phe Asn Pro 1 5 10 52 5 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 52 Met Arg Gly Ala Asn 1 5 53 87 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 53 caggaaagat ctatgagggg cgccaactaa ggggggggga aggtatttgt cccgtgcata 60 atctcgggtg ttgtctaata agaattc 87 54 4 PRT Artificial Sequence Description of Artificial Sequence N-terminal protective sequence 54 Xaa Pro Pro Xaa 1 55 36 DNA Artificial Sequence Description of Artificial Sequence primer 55 tactatagat ctatgaccaa acaggaaaaa accgcc 36 56 36 DNA Artificial Sequence Description of Artificial Sequence primer 56 tatacgtatt cagttgctca catgttcttt cctgcg 36 57 41 DNA Artificial Sequence Description of Artificial Sequence primer 57 aattcatact atagatctat gaccaaacag gaaaaaaccg c 41 58 42 DNA Artificial Sequence Description of Artificial Sequence primer 58 tatataatac atgtcagaat tcgaggtttt caccgtcatc ac 42 59 96 DNA Artificial Sequence Description of Artificial Sequence randomized oligonucloetide 59 tactatagat ctatgnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnncatag atctgcgtgc tgtgat 96 60 21 DNA Artificial Sequence Description of Artificial Sequence primer 60 atcacagcac gcagatctat g 21 61 36 DNA Artificial Sequence Description of Artificial Sequence randomized oligonucleotide 61 tactatgaat tcnnngaatt ctgccaccac tactat 36 62 21 DNA Artificial Sequence Description of Artificial Sequence primer 62 atagtagtgg tggcagaatt c 21 63 105 DNA Artificial Sequence Description of Artificial Sequence randomized oligonucleotide 63 tactatagat ctatgccgcc gnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnn nccgccgtaa taagaattcg tacat 105 64 24 DNA Artificial Sequence Description of Artificial Sequence primer 64 atgtacgaat tcttattacg gcgg 24 65 90 DNA Artificial Sequence Description of Artificial Sequence randomized oligonucleotide 65 tactatagat ctatgvanva nvanvanvan vanvanvanv anvanvanva nvanvanvan 60 vanvantaat aagaattctg ccagcactat 90 66 24 DNA Artificial Sequence Description of Artificial Sequence primer 66 atagtgctgg cagaattctt atta 24 67 105 DNA Artificial Sequence Description of Artificial Sequence randomized oligonucleotide 67 tactatagat ctatggaaga cgaagacnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnncgtaa acgtaaataa taagaattcg tacat 105 68 30 DNA Artificial Sequence Description of Artificial Sequence primer 68 atgtacgaat tcttattatt tacgtttacg 30 69 81 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 69 agatctatgc cgccgattct atggggcgaa gcgagaaagc gcttgtgggg tggggatcat 60 acaccgccgt aataagaatt c 81 70 21 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 70 Met Pro Pro Ile Leu Trp Gly Glu Ala Arg Lys Arg Leu Trp Gly Gly 1 5 10 15 Asp His Thr Pro Pro 20 71 90 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 71 agatctatgc cgccgccgtt ggatattgtg tcgggtattg aggtaggggg gcatttgtgg 60 tgccgccgta ttaagaattc tcatgtttga 90 72 27 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 72 Met Pro Pro Pro Leu Asp Ile Val Ser Gly Ile Glu Val Gly Gly His 1 5 10 15 Leu Trp Cys Arg Arg Ile Lys Asn Ser His Val 20 25 73 81 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 73 agatctatgc cgccggacaa tccggtcctg tgatgaagcg gaggtcgacc aaggggatat 60 cagccgccgt aataagaatt c 81 74 8 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 74 Met Pro Pro Asp Asn Pro Val Leu 1 5 75 81 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 75 agatctatgc cgccgctatt ggacggagat gacaaataga tatatgcgtg gttgtttttc 60 tgtccgccgt aataagaatt c 81 76 10 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 76 Met Pro Pro Leu Leu Asp Gly Asp Asp Lys 1 5 10 77 79 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 77 agatctatgc cgccgaggtg gaagatgttg ataagacagt gacagatgcg ttccattact 60 cccgccgtaa taagaattc 79 78 11 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 78 Met Pro Pro Arg Trp Lys Met Leu Ile Arg Gln 1 5 10 79 39 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 79 agatctatga tgagagtagc gccgccgtaa taagaattc 39 80 7 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 80 Met Met Arg Val Ala Pro Pro 1 5 81 81 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 81 agatctatgc cgccgttgcg cggggcatgc gatgtatatg gggtaaattg aatgtcttgt 60 gggccgccgt aataagaatt c 81 82 14 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 82 Met Pro Pro Leu Arg Gly Ala Cys Asp Val Tyr Gly Val Asn 1 5 10 83 81 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 83 agatctatgc cgccggggag aggggaagcg gtgggagtga catgcttgag cgcgaacgtg 60 tacccgccgt aataagaatt c 81 84 21 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 84 Met Pro Pro Gly Arg Gly Glu Ala Val Gly Val Thr Cys Leu Ser Ala 1 5 10 15 Asn Val Tyr Pro Pro 20 85 81 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 85 agatctatgc cgccgggaag ggtagtgttc tttgtcgcta tctttgtttc cgcaatatgc 60 ctcccgccgt aataagaatt c 81 86 21 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 86 Met Pro Pro Gly Arg Val Val Phe Phe Val Ala Ile Phe Val Ser Ala 1 5 10 15 Ile Cys Leu Pro Pro 20 87 81 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 87 agatctatgc cgccgaggtt cgctcatgag agtgttaaag ggctggggga cgttacaaaa 60 gctccgccgt aataagaatt c 81 88 21 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 88 Met Pro Pro Arg Phe Ala His Glu Ser Val Lys Gly Leu Gly Asp Val 1 5 10 15 Thr Lys Ala Pro Pro 20 89 72 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 89 agatctatgc atgacgaaca agaggaggag cacaataaaa aggataacga aaaagaacac 60 taataagaat tc 72 90 18 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 90 Met His Asp Glu Gln Glu Glu Glu His Asn Lys Lys Asp Asn Glu Lys 1 5 10 15 Glu His 91 75 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 91 agatctatgc agcaggagca cgagcaaggc aggatgagca agaggatgaa gaataataag 60 aattctcatg tttga 75 92 22 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 92 Met Gln Gln Glu His Glu Gln Gly Arg Met Ser Lys Arg Met Lys Asn 1 5 10 15 Asn Lys Asn Ser His Val 20 93 75 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 93 agatctatga accatcataa tgaggccatg atcaacacaa tgaaaacgag gaataataag 60 aattctcatg tttga 75 94 22 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 94 Met Asn His His Asn Glu Ala Met Ile Asn Thr Met Lys Thr Arg Asn 1 5 10 15 Asn Lys Asn Ser His Val 20 95 72 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 95 agatctatga acgacgacaa tcagcaagag gataatcatg atcagcataa ggataacaaa 60 taataagaat tc 72 96 18 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 96 Met Asn Asp Asp Asn Gln Gln Glu Asp Asn His Asp Gln His Lys Asp 1 5 10 15 Asn Lys 97 72 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 97 agatctatgc aagagcagga tcagcataat gataaccatc acgaggataa acataagaag 60 taataagaat tc 72 98 18 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 98 Met Gln Glu Gln Asp Gln His Asn Asp Asn His His Glu Asp Lys His 1 5 10 15 Lys Lys 99 93 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 99 agatctatgg aagacgaaga cgagggtgcg tcagcgtggg gagcagaact ttggtcgtgg 60 cagtcggtgc gtaaacgtaa ataataagaa ttc 93 100 25 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 100 Met Glu Asp Glu Asp Glu Gly Ala Ser Ala Trp Gly Ala Glu Leu Trp 1 5 10 15 Ser Trp Gln Ser Val Arg Lys Arg Lys 20 25 101 93 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 101 agatctatgg aagacgaaga cggtctaggc atggggggtg ggttggtcag gctcacttta 60 ttattcttcc gtaaacgtaa ataataagaa ttc 93 102 25 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 102 Met Glu Asp Glu Asp Gly Leu Gly Met Gly Gly Gly Leu Val Arg Leu 1 5 10 15 Thr Leu Leu Phe Phe Arg Lys Arg Lys 20 25 103 93 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 103 agatctatgg aagacgaaga cggggagagg atccaggggg cccgctgtcc agtagcgctg 60 gtagatagac gtaaacgtaa ataataagaa ttc 93 104 25 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 104 Met Glu Asp Glu Asp Gly Glu Arg Ile Gln Gly Ala Arg Cys Pro Val 1 5 10 15 Ala Leu Val Asp Arg Arg Lys Arg Lys 20 25 105 11 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 105 Met Glu Asp Glu Asp Asp Arg Gly Arg Gly Arg 1 5 10 106 93 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 106 agatctatgg aagacgaaga cgacaggggg cgtgggcggt agctttaagt tgcgctaagt 60 tgcgagatac gtaaacgtaa ataataagaa ttc 93 107 93 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 107 agatctatgg aagacgaaga cgggggggcc gggaggaggg cctgtctttg ttccgcgctt 60 gttggggaac gtaaacgtaa ataataagaa ttc 93 108 25 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 108 Met Glu Asp Glu Asp Gly Gly Ala Gly Arg Arg Ala Cys Leu Cys Ser 1 5 10 15 Ala Leu Val Gly Glu Arg Lys Arg Lys 20 25 109 90 DNA Artificial Sequence Description of Artificial Sequence nucleic acid encoding stabilized peptide 109 agatctatgg aagacgaaga caagcgtcgc gagaggagtg caaaagggcg tcatgtcggt 60 cggtcgatgc gtaaacgtaa ataagactgt 90 110 25 PRT Artificial Sequence Description of Artificial Sequence stabilized peptide 110 Met Glu Asp Glu Asp Lys Arg Arg Glu Arg Ser Ala Lys Gly Arg His 1 5 10 15 Val Gly Arg Ser Met Arg Lys Arg Lys 20 25 

What is claimed:
 1. A method for making a non-naturally occurring polypeptide, the method comprising: attaching a first stabilizing group to the N-terminus of a bioactive peptide; and attaching a second stabilizing group to the C-terminus of the bioactive peptide to yield the non-naturally occurring polypeptide; wherein the first stabilizing group is selected from the group consisting of a small stable protein, Pro-, Pro-Pro-, Xaa-Pro- and Xaa-Pro-Pro-, and wherein the second stabilizing group is selected from the group consisting of a small stable protein, -Pro, -Pro-Pro, -Pro-Xaa and -Pro-Pro-Xaa.
 2. The method of claim 1 wherein the non-naturally occurring polypeptide is chemically synthesized.
 3. The method of claim 1 wherein attaching either or both of the first and second stabilizing groups to the bioactive peptide comprises the following steps in the order recited: (a) providing the bioactive peptide; and (b) attaching either or both of the first and second stabilizing groups to the bioactive peptide.
 4. The method of claim 1 wherein either or both of the first and second stabilizing groups is attached to the bioactive peptide during synthesis of the bioactive peptide.
 5. The method of claim 1 wherein the stabilizing group is isolated from an endogenous source, chemically synthesized, enzymatically synthesized, or produced using recombinant DNA technology.
 6. The method of claim 1 further comprising determining the stability of the non-naturally occurring polypeptide relative to the bioactive peptide.
 7. A method for making a non-naturally occurring polypeptide, the method comprising: providing a host cell transformed with a nucleic acid encoding a non-naturally occurring polypeptide, the non-naturally occurring polypeptide comprising a bioactive peptide, a first stabilizing group attached to the N-terminus of the bioactive peptide, and a second stabilizing group attached to the C-terminus of the bioactive peptide, wherein the first stabilizing group is selected from the group consisting of a small stable protein, Pro-, Pro-Pro-, Xaa-Pro- and Xaa-Pro-Pro-, and wherein the second stabilizing group is selected from the group consisting of a small stable protein, -Pro, -Pro-Pro, -Pro-Xaa and -Pro-Pro-Xaa; expressing the non-naturally occurring polypeptide in the host cell; and recovering non-naturally occurring polypeptide from the host cell.
 8. The method of claim 7 wherein the host cell is bacterial cell.
 9. The method of claim 7 wherein the host cell is a eukaryotic cell.
 10. The method of claim 1 or 7 wherein at least one of the first and second stabilizing groups comprises a small stable protein.
 11. The method of claim 1 or 7 wherein the small stable protein is a four-helix bundle protein.
 12. The method of claim 1 or 7 wherein the small stable protein is selected from the group consisting of Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, and glutathione reductase.
 13. The method of claim 1 or 7 wherein the small stable protein is Rop protein.
 14. The method of claim 1 or 7 wherein the first stabilizing group is Pro-Pro- and the second stabilizing group is -Pro-Pro.
 15. The method of claim 1 or 7 wherein the non-naturally occurring polypeptide further comprises a cleavage site immediately preceding the first stabilizing group.
 16. The method of claim 1 or 7 wherein the non-naturally occurring polypeptide further comprises a cleavage site immediately following the second stabilizing group
 17. The method of claim 1 or 7 wherein the non-naturally occurring polypeptide is an antimicrobial peptide.
 18. The method of claim 1 or 7 wherein the non-naturally occurring polypeptide is a therapeutic peptide drug.
 19. The method of claim 1 or 7 wherein the bioactive peptide is a naturally occurring bioactive peptide.
 20. A method for making a non-naturally occurring polypeptide, the method comprising: attaching a stabilizing group to either or both of the N-terminus or C-terminus of a bioactive peptide; wherein the stabilizing group attached to the N-terminus comprises Xaa-Pro-Pro-, and the stabilizing group attached to the C-terminus comprises -Pro-Pro-Xaa.
 21. A method for making a non-naturally occurring polypeptide, the method comprising: attaching a stabilizing group comprising Rop protein to either or both of the N-terminus or C-terminus of the bioactive peptide.
 22. A method for making a non-naturally occurring polypeptide, the method comprising: attaching a stabilizing group comprising a four-helix bundle protein to either or both of the N-terminus or C-terminus of the bioactive peptide.
 23. The method of any of claims 20, 21 and 22 wherein the non-naturally occurring polypeptide is chemically synthesized.
 24. The method of any of claims 20, 21 and 22 wherein attaching the stabilizing group to the bioactive peptide comprises the following steps in the order recited: (a) providing the bioactive peptide; and (b) attaching the stabilizing group to the bioactive peptide.
 25. The method of any of claims 20, 21 and 22 wherein the stabilizing group is attached to the bioactive peptide during synthesis of the bioactive peptide.
 26. The method of any of claims 20, 21 and 22 wherein the stabilizing group is isolated from an endogenous source, chemically synthesized, enzymatically synthesized, or produced using recombinant DNA technology.
 27. The method of any of claims 20, 21 and 22 further comprising determining the stability of the non-naturally occurring polypeptide relative to the bioactive peptide.
 28. A method for making a non-naturally occurring polypeptide, the method comprising: providing a host cell transformed with a nucleic acid encoding a non-naturally occurring polypeptide comprising a bioactive peptide and a stabilizing group attached to either or both of the N-terminus or the C-terminus of the bioactive peptide, wherein the stabilizing group attached to the N-terminus comprises Xaa-Pro-Pro-, and the stabilizing group attached to the C-terminus comprises -Pro-Pro-Xaa; expressing the non-naturally occurring polypeptide in the host cell; and recovering non-naturally occurring polypeptide from the host cell.
 29. A method for making a non-naturally occurring polypeptide, the method comprising: providing a host cell transformed with a nucleic acid encoding a non-naturally occurring polypeptide comprising a bioactive peptide and a stabilizing group comprising Rop protein attached to either or both of the N-terminus or the C-terminus of the bioactive peptide; expressing the non-naturally occurring polypeptide in the host cell; and recovering non-naturally occurring polypeptide from the host cell.
 30. A method for making a non-naturally occurring polypeptide, the method comprising: providing a host cell transformed with a nucleic acid encoding a non-naturally occurring polypeptide comprising a bioactive peptide and a stabilizing group comprising a four-helix bundle protein attached to either or both of the N-terminus or the C-terminus of the bioactive peptide; expressing the non-naturally occurring polypeptide in the host cell; and recovering non-naturally occurring polypeptide from the host cell.
 31. The method of any of claims 28, 29 and 30 wherein the host cell is bacterial cell.
 32. The method of any of claims 28, 29 and 30 wherein the host cell is a eukaryotic cell.
 33. The method of any of claims 28, 29 and 30 wherein the non-naturally occurring polypeptide is an antimicrobial peptide.
 34. The method of any of claims 28, 29 and 30 wherein the non-naturally occurring polypeptide is a therapeutic peptide drug.
 35. The method of any of claims 28, 29 and 30 wherein the bioactive peptide is a naturally occurring bioactive peptide. 